Antibodies to GM-CSF

ABSTRACT

Hybridoma lines that secrete human monoclonal antibodies with high binding specificity and biological activity, particularly neutralizing activity against granulocyte-macrophage colony stimulating factor, and methods of generating the hybridoma lines are provided. Target antigens and epitopes are also provided. The antibodies may be used in therapeutic methods, for example in the treatment of cancer, infectious disease, or autoimmune disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit to U.S. Provisional Patent ApplicationNos. 60/771,251 filed Feb. 8, 2006 and 60/774,500 filed Feb. 17, 2006,the contents of each of which are incorporated by reference herein, intheir entirety.

FIELD OF THE INVENTION

The invention relates generally to the field of immunotherapeutics. Morespecifically, the invention relates to antigens for generatingmonoclonal antibodies, and monoclonal antibodies that can neutralizeautoimmune and cancer cells, and can neutralize the inflammatoryresponse.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications,technical articles and scholarly articles are cited throughout thespecification. Each of these cited publications is incorporated byreference herein, in its entirety and for all purposes

Several disease-associated antigens are currently being targeted usingtherapeutic monoclonal antibodies (MAbs) because of their uniquepharmacological and safety profiles. Among the disease-associated targetantigens are CD20, Tumor Necrosis Factor alpha (TNF-α), Epidermal GrowthFactor Receptors (EGFR), and granulocyte-macrophage colony stimulatingfactor.

Granulocyte macrophage-colony stimulating factor (GM-CSF) was originallydiscovered as a protein with the capacity to generate both granulocyteand macrophage colonies from precursor cells in mouse bone marrow, andwas accordingly named (Burgess et al. (1980) Blood 56:947-58).Subsequent studies have demonstrated a role of GM-CSF in potentiatingthe function of mature macrophages and granulocytes (Handman and Burgess(1979) J. Immunol. 122:1134-1137; Hamilton et al. (1980) J. CellPhysiol. 103:435-445; Gamble et al. (1985) Proc. Natl. Acad. Sci. USA82:8667-8671), suggesting a role for GM-CSF in inflammatory responses(Hamilton et al. (1980) J. Cell Physiol. 103:435-445). As the moleculewas studied it became clear that GM-CSF has other functions arising fromits ability to affect the properties of more mature myeloid cells suchas granulocytes, macrophages and eosinophils. The functions of GM-CSFare mediated by binding to CD116, the granulocyte-macrophage colonystimulating factor receptor, also known as colony stimulating factor 2receptor alpha that binds GM-CSF with low affinity. The beta subunit,called CD131, which is also shared with the IL3 and IL5 receptors, hasno detectable binding activity for GM-CSF by itself but is necessary forhigh affinity binding when in association with the alpha subunit andplays a fundamental role in signal transduction. The GM-CSF receptorsare found on myeloid progenitors and mature myeloid cells includingneutrophils, eosinophils, mononuclear phagocytes, and monocytes. Inaddition, GM-CSF receptor subunits have been shown to be present innormal, non-hematopoietic tissues such as human placenta, endothelium,and oligodendrocytes of the central nervous system.

GM-CSF plays a major biological role in the generation of granulocytesand macrophages from early bone marrow progenitors within the bonemarrow. What was not appreciated at first but later uncovered wasadditional physiological functions of GM-CSF in host responses toexternal stimuli and in inflammatory and autoimmune conditions. In veryearly studies, GM-CSF was purified from lung tissue-conditioned mediumfollowing lipopolysaccharide (LPS) injection into mice (Burgess et al.(1977) J. Biol. Chem. 252:1998-2003). GM-CSF is considered by manyinvestigators to be one of the major regulators of granulocyte,macrophage and eosinophil lineage cell number and activation state undernormal physiological conditions. However, it has also been hypothesizedthat aberrant expression of GM-CSF may lead to altered immune andinflammatory responses with associated pathologic consequences. It wassuggested several years ago that GM-CSF should be viewed as aproinflammatory cytokine (Hamilton et al., 1980, J. Cell Physiol.103:435-445). Furthermore, GM-CSF may play a role in the diathesis of amultitude of human inflammatory pathologies, such as rheumatoidarthritis, autoimmune pathologies, inflammatory renal disease andinflammatory lung disorders such as asthma and chronic obstructivepulmonary disease (COPD). Interestingly, it has been proposed that thereis a link between multiple sclerosis and GM-CSF (McQualter et al. (2001)J. Exp. Med., 194:873-881). In an experimental model of autoimmuneencephalomyelitis, a model for multiple sclerosis, GM-CSF was found tobe involved in the autoimmune-mediated demyelination.

In vivo studies following monocyte, macrophage and neutrophil treatmentwith GM-CSF have demonstrated that GM-CSF can activate these cell typesand prolong their survival characteristics. Moreover, GM-CSF exposureresults in release of inflammatory mediators from these cell types, andfurther studies have demonstrated the ability of these cells to killcertain organisms and even tumor cells (Hamilton (1993) Immunol. Today14:18-24; Hamilton, (1993) Lancet 342:536-539; Takahashi, (1993) Blood81:357-364). To determine if the in vivo studies were indicative of thefunction of GM-CSF in vivo, systemic administration was performed withrodents. It was shown that artificially increasing circulating levels ofGM-CSF by intraperitoneal administration of the protein did result inincreased numbers of both circulating neutrophils and cycling peritonealmacrophages and that there was an increase in the development anddifferentiation of CD5+ macrophages in the peritoneal cavity of rodents(Metcalf et al., (1987) Exp. Hematol. 15:1-9).

It has also been shown that GM-CSF can “prime” cells to respond in amore robust, synergistic manner to a second stimulus, such as LPS orinterferon-gamma (Hart et al., 1988, J. Immunol. 141:1516-1521). Micecan be primed both in vitro as well as in vivo with GM-CSF so that theyproduce increased levels of circulating pro-inflammatory cytokinesfollowing subsequent challenge with LPS or TNF-alpha.

In a clinical setting, administration of GM-CSF into peritoneal dialysispatients resulted in a marked recruitment of macrophages (Selgas et al.,1996, Kidney Int. 50:2070-2078). Interestingly, and as predicted fromthe rodent studies, administration of GM-CSF in a clinical setting canresult in accentuated production of inflammatory cytokines and potentialunwanted side effects. For example, when patients with rheumatoidarthritis were treated with GM-CSF to correct the neutropenia associatedwith Felty's syndrome, their arthritis was exacerbated (Hazenberg etal., 1991, Blood 74:2769-2770). In another clinical setting, followingcancer chemotherapy, GM-CSF treatment made rheumatoid arthritis worse(de Vries et al., (1991) J. Immunol. 163: 4985-4993). Systemicadministration of GM-CSF to human donors increased the ability ofisolated granulocytes to produce superoxide, and both accentuated thecytotoxicity of circulating monocytes as well as led to an increase inthe number of monocytes (Perkins et al., 1993, Am J. Hematol.43:279-285). Aberrant expression of GM-CSF is associated with disease ofthe lung in human as well. For example, it appears that upregulation ofGM-CSF in the lung by minor irritants, endotoxins or infectionspredisposes towards TH2 immune deviation and asthma (Eisenbarth et al.(2002) J. Exp. Med. 196:1645-1651). The studies summarized above suggestthat GM-CSF plays a role in the activation of the inflammatory processthrough cell recruitment, increased cell survival and/or priming foractivation.

Several association and experimental data suggest a role for GM-CSF inasthma. The use of neutralizing antibodies in a mouse model of asthmahave demonstrated the ability to suppress asthmatic phenotypes(Yamashita (2002) Cell Immunol. 219:92), while several studies measuringcytokines in BAL fluid of asthmatic patients have found an increase inGM-CSF (Gajewska (2003) Curr Drug Targets Inflamm Allergy 2:279).

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune diseasefor which there is ample evidence that GM-CSF may be involved. GM-CSFhas been found at elevated levels in RA lesions (Xu et al. (1989) J.Clin. Invest. 83:876) and is produced in vitro by resident joint cells(chondrocytes and synovial fibroblasts) following their stimulation withinflammatory cytokines such as IL-1 and TNF-alpha (Leizer et al. (1990)Blood 76:1989). Collagen-induced arthritis (CIA) in the mouse is anautoimmune model of RA that is dependent upon both humoral and cellularimmune responses to type II collagen (CII) (Seki et al. (1988) J.Immunol. 140, 1477). Historically this RA phenotype is restricted tomouse strains bearing the H-2q or H-2r haplotypes and is generallyperformed in DBA/1 mice (Wooley (1988) Methods Enzymol. 162:361). Aseries of studies were performed in transgenic mice that were homozygousnull for the murine GM-CSF locus (Stanley et al. (1994) Proc. Natl.Acad. Sci. USA 91:5592). Interestingly, the GM-CSF-deficient mice areresistant to the induction of collagen-induced arthritis as compared totheir wild-type control litter mates (Campbell et al. (1998) J. Immunol.161:3639-3644).

Of further interest is that GM-CSF null mice have impaired surfactantclearance that leads to murine pulmonary alveolar proteinosis (PAP),which closely mimics the human condition as described herein. Moreover,the PAP phenotype can be corrected by lung-specific delivery of theGM-CSF gene (Zsengaller et al. (1998) Hum. Gene Ther. 9:2101-2109),aerosolization of GM-CSF or bone marrow transplantation forhematopoeitic reconstitution (Reed et al. (1999) Am. J. Physiol.276:L556-L563; Nishinakamura et al. (1996) J. Exp. Med. 183:2657-2662).

Adult human pulmonary alveolar proteinosis (PAP) is a rare diseasecharacterized by the accumulation of phospholipids and surfactantproteins in the alveoli. It has been hypothesized that PAP is due to theinability of the alveolar macrophages and type II epithelial cells toclear excess surfactant (Mazzone et al. (2001) Clev. Clin. J. Med.68:977-992). The diagnosis of PAP generally requires an open lung biopsyand the standard therapy for the disease is physical removal of theaccumulated surfactant by whole-lung lavage (Shah et al. (2000) Thorax55:67-77). Furthermore, patients with PAP have been shown to havecirculating, neutralizing antibodies to GM-CSF, thereby implicating thiscytokine as causative of the disease. Whether this autoimmune responseis specific for GM-CSF is unclear. However, it has been shown that asubset of PAP patients improve with GM-CSF therapy, supporting thehypothesis that the absence of GM-CSF either by gene disruption orantibody-mediated neutralization results in the development of PAP.

There is also evidence to support a role for GM-CSF in cancer. Forexample, GM-CSF plays a role in the genesis and progression ofleukemias, such as juvenile myelomonocytic leukemia (JMML); Emanuel P D(2004) Curr. Hematol. Rep. 3:203-209). JMML is characterized bydisruption of normal haemopoiesis resulting in excessive, inappropriateproliferation of immature myeloid cells in the bone marrow. Theseproliferating hematopoietic cancer cells can metastasize to the spleenand liver. Interestingly, patients with JMML are hypersensitive toGM-CSF and exhibit pathologic features similar to those in transgenicmice that over-express GM-CSF (Lang et al. (1987) 51:675-86).Furthermore, GM-CSF has been shown to promote JMML cell growth andsurvival (Emanuel et al (1991) Blood 77:925-9). In the transgenic mousemodel of JMML, blockade of GM-CSF reduced JMML cell burden in the bonemarrow, blood and spleen (Iversen et al. (1997) Blood, 90:4910-7).

It is clear from murine disease models in which GM-CSF is knocked-out aswell as human diseases such as PAP in which circulating antibodies aregenerated in the systemic circulation to GM-CSF that this cytokine is animportant mediator of pathology. Therefore an approach to develop a drugthat can antagonize the activity of GM-CSF, either by developing anantibody to the cytokine itself or by blockade of the GM-CSF receptor,may be a valuable human therapeutic. Several polyclonal and monoclonalantibodies have been generated to the recombinant GM-CSF molecule. Forexample, Beffy et al. ((1994), Hybridoma 13:457-468), generatedpolyclonal antibodies to recombinant human GM-CSF in New Zealand Whiterabbits and monoclonal antibodies in Balb/c mice. These rabbit and someof the murine monoclonal antibodies were capable of neutralizing theactivity of GM-CSF in an in vitro cell proliferation assay with MO7ccells. In further studies, Nice et al. (1990, Growth Factors 3, 159-169)epitope-mapped the binding site of one neutralizing murine anti-GM-CSFantibody, LMM102. A well-defined epitope was delineated by generating aseries of digestion products from recombinant, human GM-CSF, usingreverse phase HPLC fractionation to separate the fragments, followed byadditional S. aureus V8 digestion of the tryptic fragments to define aproduct comprising two peptides linked by a disulphide bond. Threemurine antibodies to human GM-CSF were generated by Dempsey et al.(1990, Hybridoma 9, 545-558) that neutralized GM-CSF in an in vitroassay system with an EC50 in the 0.1 to 1.7 nanomolar range. Theseantibodies did not react with murine GM-CSF or other related cytokines.All of the above-described antibodies are useful reagents for thedetection of GM-CSF in human serum as well as for in vitro assays toinhibit GM-CSF signaling. However, all of these antibodies have littlevalue as therapeutics due to the fact that they are derived from eithera murine or rabbit system. Attempts have been made to generate chimericantibodies from murine counterparts by subcloning the variable domainfrom the murine anti-GM-CSF antibody into a human backbone. Thisstrategy has led to a chimeric antibody that can neutralize GM-CSF invitro and may be useful as a therapeutic (WO 03/068924 A2).

An important aspect of a therapeutic antibody is its ability to elicitimmune effector functions, such as antibody dependent cellularcytotoxicity (ADCC). Rodent MAbs, for example, have been shown to poorlymediate effector functions in humans because of sequence differences inthe Fc region and therefore chimerization or humanization are requiredto gain optimal pharmacological properties. In addition, MAbs with fullyhuman sequences may still fail to support ADCC if they are produced innon-human host cells that may alter native glycosylation pattern of MAbs(Shinkawa et al. (2003) J. Biol. Chem. 278:3466-73).

In view of these facts, production of therapeutic antibodies by humanB-cells is preferred. Methods for generation of hybridomas secretinghuman MAbs have been previously reported (WO2004/046330). TherapeuticMAbs generated by human B-cells are able to exert human effectorfunctions and have very limited immunogenicity because of their nativehuman structure. The generation of hybridoma or Epstein-Barr virus(EBV)-transformed lymphoblastoid lines derived from human B-cells hasbeen previously reported (Kirman et al. (2002) Hybrid Hybridomics21:405-14; Boerner et al. (1991) J. Immunol. 147:86-95; Zafiropoulos etal. (1997) J. Immunol. Methods 200:181-90); however, information on thecharacterization of these antibodies and the lines with respect to theirlong term stability, suitability to manufacturing processes, and theantibody's pharmacological properties is limited (van Dijk et al. (2001)Curr. Opin. Chem. Biol. 5:368-74).

There is thus a need for therapeutic human antibodies for the treatmentof inflammation associated with infectious, inflammatory diseases,autoimmune disorders, and other diseases such as cancer. It is furtherdesired that such antibodies would elicit immune effector functions, aswell as be well-tolerated in human patients. The present inventionaddresses these and other long felt needs.

SUMMARY OF THE INVENTION

The invention features isolated human antibodies that specifically bindto GM-CSF. The antibodies can comprise a heavy chain CDR3 having SEQ IDNO:42 or 48. In some preferred embodiments, the antibodies can comprisea heavy chain having SEQ ID NO:8 or 16. In some preferred embodiments,the antibodies can comprise a light chain having SEQ ID NO:12 or 18. Insome embodiments, the antibodies have two heavy chains. In someembodiments, the antibodies have two light chains. The antibodies arehigh affinity antibodies, and can have an affinity of less than about1×10⁻⁸ M. Preferably, the antibodies are monoclonal antibodies, and morepreferably, are human monoclonal antibodies. In highly preferredembodiments, the antibodies specifically bind to an epitope on apolypeptide comprising the amino acid sequence SEQ ID NO:3, 4, 5, 35,36, 37, 38, or 39. Cells that express such antibodies, such as hybridomacells are also provided.

The invention also features polynucleotides that encode antibodies thatspecifically bind to GM-CSF. In some preferred embodiments, thepolynucleotides comprise a heavy chain sequence of SEQ ID NO:10 or 17.In some preferred embodiments, the polynucleotides comprise a lightchain sequence of SEQ ID NO:14 or 19. Vectors comprising suchpolynucleotides are also provided.

The invention also features methods for treating GM-CSF-mediatedinflammatory disease in a subject in need of such treatment. The methodscomprise administering to the subject a composition comprising apharmaceutically acceptable carrier and at least one antibody thatspecifically binds to GM-CSF in an amount effective to treat aGM-CSF-mediated inflammatory disease. In preferred aspects of thesemethods, the antibodies can comprise a heavy chain CDR3 having SEQ IDNO:42 or 48. In some preferred embodiments, the antibodies can comprisea heavy chain having SEQ ID NO:8 or 16. In some preferred embodiments,the antibodies can comprise a light chain having SEQ ID NO:12 or 18. Theantibodies are high affinity antibodies, and can have an affinity ofless than about 1×10⁻⁸ M.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the antigen panel ELISA for selection ofantigen-specific human MAbs. Three GM-CSF-specific human MAbs (E5, G7,E10), reacted with human GM-CSF and none of the other antigens in thepanel. Antibody 215 is a murine MAb that binds to human GM-CSF (hGM-CSF)and murine GM-CSF (mGM-CSF).

FIGS. 2A and 2B illustrate the high specificity of human MAbs to nativehuman GM-CSF. As shown in FIG. 2A, soluble human GM-CSF was bound tomurine hybridoma cells presenting cell surface immunoglobulin (Ig) tohuman GM-CSF. MAb E5 was subsequently added to the reaction and itsbinding to human GM-CSF measured using FITC-conjugated goat anti-humanIg. E5 did not bind any of the surface proteins expressed by the murinehybridoma cells (middle panel) but only bound soluble GM-CSF captured bythe cell surface Ig (lower panel). As shown in FIG. 2B, phycoerythrin(PE)-labeled human GM-CSF (PE-GM) can react to Ig expressed on E10 cellsurface. Excess of unlabeled GM-CSF (bottom panel) competed for PE-GMbinding.

FIG. 3 demonstrates secretion of antigen-binding IgG by class-switchedhybridoma cells. Hybridoma E5 cells (parent) were treated as describedin Materials and Methods. Hybridoma clones that had class-switched(switched) were identified using an ELISPOT-based screening method. AnELISA measuring specific binding to human GM-CSF coated onto plates wascarried out to assess binding of either IgM or IgG. Switched IgG MAbsexhibited comparable binding to antigen as the parental IgM.

FIG. 4 demonstrates GM-CSF-dependent growth inhibition of TF-1 celllines with fully human MAbs. The human GM-CSF-dependent humanerythroleukemia cell line TF-1 (ATCC, VA) was grown in complete RPMI1640 containing 10 ng/mL recombinant human GM-CSF (PeproTech, NJ). Onthe day preceding the experiment, TF-1 cells were grown in 0.1% FBS inthe absence of GM-CSF. The starved TF-1 cells were harvested and washedtwice with assay medium (plain RPMI with 0.5% BSA). Cells wereresuspended in assay medium and seeded in 96-well microplates at aconcentration of 10,000 cell/well. Wells contained either assay medium,100 pg/mL GM-CSF, or GM-CSF pre-incubated for one hour with test orisotype control Igs at concentrations indicated in the figure. After 3days, 40 μL of Cell Titer reagent (Promega, WI) was added to each well,and plates were further incubated at 37° C. for 1 hour. Optical density(O.D.) was measured at 490 nm in spectrophotometer and medium backgroundwas subtracted from samples. Percentage of GM-CSF neutralization wascalculated as follows: 100−[O.D. with Ig/O.D. without Ig)×100].

FIG. 5A illustrates ELISA results demonstrating that all E5-3D2subclones tested secrete high levels of Ig. The hybridoma E5-3D2 linewas grown for 60 generations and then stability of production wasassessed by analyzing frequency of producing cells. Subclones (X1-X10)derived from 3D2 cells via limiting dilution were randomly chosen andtheir Ig production measured using an ELISA-based assay. Absorbance at405 nm was normalized for colony size by visual inspection of thecell-containing wells. FIG. 5B demonstrates that the specificproductivity measured during the log phase was 24 pg/cell/day followinginoculation of 3D2 cells in a stirred bioreactor containing 1 liter ofserum-free medium and Ig production and recordation of number of viablecells on day 1 through 5.

FIG. 6A shows results of an example of a single nucleotide deletion inthe BAT marker found in E5 hybridoma cells treated with mismatch repairinhibitors. Dotted lines crossing the central peak in the histogramrepresent the size of wild type (wt) or contracted (−1 nt) fragment. InFIG. 6B, parental and mismatch repair-inhibited cells were seeded inmicroplates to yield 3,763 and 2,437 Ig-secreting clones (O.D.>0.2),respectively. Ig concentrations were determined by ELISA and thefrequency of clones with O.D. values greater than 1 was recorded andexpressed as percentage of total number of clones screened.

FIG. 7 provides a diagrammatic flow chart describing each step for thegeneration of a fully human hybridoma cell line.

FIG. 8 illustrates an antigen panel ELISA for selection ofantigen-specific human MAbs. Three GM-CSF specific huMAbs, E10, G9, andE5 (not described), reacted with human GM-CSF and none of the otherantigens in the panel. Efficient antigen coating was optimized usingspecific MAbs to the various antigens for greater assay sensitivity. 615and 215 are two anti-human GM-CSF murine MAbs.

FIG. 9 demonstrates isotype determination of MAb E10. To determine theisotype of E10, a standard analysis was performed using anti-human IgG,IgG1, IgG2, IgG3, IgM, Lκ, and Lλ Fc specific antibodies to derive theisotype.

FIG. 10 shows a Western analysis with E10 and G9 MAbs. The western blotanalyses were performed with E10 and G9 to determine if the antibodieswould cross-react with human recombinant GM-CSF. The mAb215 is mouseanti-human GM-CSF neutralizing monoclonal antibody as a positivecontrol, Lane: 1 were loaded with tumor cell lysate as negative proteincontrol, Lane:2 were loaded with 500 ng rh GM-CSF (PeProTech, NJ USA),As shown in FIG. 5 E10 and G9 reacted with human GM-CSF.

FIG. 11 shows a BIACORE analysis for E10. E10.20 had an association rateconstant (k_(a))=2.47×10⁴, a dissociation rate constant(k_(d))=2.16×10⁻⁵ and an overall affinity (K_(D)) of 0.87 nM.

FIG. 12 demonstrates isotype determination of MAb G9. To determine theisotype of G9, a standard analysis was performed using anti-human IgG,IgG1, IgG2, IgG3, IgM, Lκ, and Lλ specific antibodies to derive theisotype (in double samples, FIG. 3).

FIG. 13 shows a BIACORE analysis for G9. 10G9.1 had an association rateconstant (k_(a))=8.47×10⁶, a dissociation rate constant(k_(d))=9.27×10⁻⁵ and an overall affinity (K_(D)) of 0.87 nM.

FIG. 14 illustrates a strategy to map G9 epitope. Overlapping peptidesused to map G9 epitope binding location correspond to amino acidsequences of SEQ ID NOs: 35-38, and 64-85.

FIG. 15 shows G9 epitope mapping analysis using Western blot analysiswith overlapping peptides that cover human GM-CSF protein sequencecorresponding to amino acid sequences of SEQ ID NOs: 35-38, and 65-86.Antibody positive peptides include SEQ ID NO: 35-38.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Various terms relating to the methods and other aspects of the presentinvention are used throughout the specification and claims. Such termsare to be given their ordinary meaning in the art unless otherwiseindicated. Other specifically defined terms are to be construed in amanner consistent with the definition provided herein.

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting. As used in this specificationand the appended claims, the singular forms “a”, “an” and “the” includeplural referents unless the content clearly dictates otherwise. Thus,for example, reference to “a cell” includes a combination of two or morecells, and the like.

Each range recited herein includes all combinations and sub-combinationsof ranges, as well as specific numerals contained therein.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or 10%, more preferably ±5%, even morepreferably +1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

“Infectious disease” includes, but is not limited to, infection with apathogen, virus, bacterium, fungus or parasite. Examples of virusesinclude, but are not limited to, severe acute respiratory syndrome(SARS; caused by SARS-associated coronavirus), hepatitis type B or typeC, influenza, varicella, adenovirus, herpes simplex virus type I or typeII, rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytialvirus, papilloma virus, papova virus, cytomegalovirus, echinovirus,arbovirus, hantavirus, coxsachie virus, mumps virus, measles virus,rubella virus, polio virus, and human immunodeficiency virus (HIV) typeI or type II. Examples of bacteria include, but are not limited to,Ebola, Staphylococcus A-E, Plasmodium (malaria), M. tuberculosis,mycobacterium, mycoplasma, neisseria and legionella. Examples ofparasites include, but are not limited to, rickettsia and chlamydia.

“Inflammatory diseases” include, but are not limited to, acute andchronic immune and autoimmune pathologies, such as, but not limited to,rheumatoid arthritis, autoimmune disease, inflammatory renal disease andinflammatory lung disorders such as asthma and chronic obstructivepulmonary disease (COPD), multiple sclerosis, and autoimmuneencephalomyelitis.

An “autoimmune disease” is a disease or disorder arising from anddirected against an individual's own tissues or a co-segregate ormanifestation thereof or resulting condition therefrom. Examples ofautoimmune diseases include, but are not limited to arthritis(rheumatoid arthritis such as acute arthritis, chronic rheumatoidarthritis, gout or gouty arthritis, acute gouty arthritis, acuteimmunological arthritis, chronic inflammatory arthritis, degenerativearthritis, type II collagen-induced arthritis, infectious arthritis,Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still'sdisease, vertebral arthritis, and juvenile-onset rheumatoid arthritis,osteoarthritis, arthritis chronica progrediente, arthritis deformans,polyarthritis chronica primaria, reactive arthritis, and ankylosingspondylitis), inflammatory hyperproliferative skin diseases, psoriasissuch as plaque psoriasis, gutatte psoriasis, pustular psoriasis, andpsoriasis of the nails, atopy including atopic diseases such as hayfever and Job's syndrome, dermatitis including contact dermatitis,chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis,allergic contact dermatitis, dermatitis herpetiformis, nummulardermatitis, seborrheic dermatitis, non-specific dermatitis, primaryirritant contact dermatitis, and atopic dermatitis, x-linked hyper IgMsyndrome, allergic intraocular inflammatory diseases, urticaria such aschronic allergic urticaria and chronic idiopathic urticaria, includingchronic autoimmune urticaria, myositis, polymyositis/dermatomyositis,juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma(including systemic scleroderma), sclerosis such as systemic sclerosis,multiple sclerosis (MS) such as spino-optical MS, primary progressive MS(PPMS), and relapsing remitting MS (RRMS), progressive systemicsclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata,ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease(IBD) (for example, Crohn's disease, autoimmune-mediatedgastrointestinal diseases, colitis such as ulcerative colitis, colitisulcerosa, microscopic colitis, collagenous colitis, colitis polyposa,necrotizing enterocolitis, and transmural colitis, and autoimmuneinflammatory bowel disease), bowel inflammation, pyoderma gangrenosum,erythema nodosum, primary sclerosing cholangitis, respiratory distresssyndrome, including adult or acute respiratory distress syndrome (ARDS),meningitis, inflammation of all or part of the uvea, iritis,choroiditis, an autoimmune hematological disorder, rheumatoidspondylitis, rheumatoid synovitis, hereditary angioedema, cranial nervedamage as in meningitis, herpes gestationis, pemphigoid gestationis,pruritis scroti, autoimmune premature ovarian failure, sudden hearingloss due to an autoimmune condition, IgE-mediated diseases such asanaphylaxis and allergic and atopic rhinitis, encephalitis such asRasmussen's encephalitis and limbic and/or brainstem encephalitis,uveitis, such as anterior uveitis, acute anterior uveitis, granulomatousuveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterioruveitis, or autoimmune uveitis, glomerulonephritis (GN) with and withoutnephrotic syndrome such as chronic or acute glomerulonephritis such asprimary GN, immune-mediated GN, membranous GN (membranous nephropathy),idiopathic membranous GN or idiopathic membranous nephropathy, membrano-or membranous proliferative GN (MPGN), including Type I and Type II, andrapidly progressive GN, proliferative nephritis, autoimmunepolyglandular endocrine failure, balanitis including balanitiscircumscripta plasmacellularis, balanoposthitis, erythema annularecentrifugum, erythema dyschromicum perstans, eythema multiform,granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus,lichen simplex chronicus, lichen spinulosus, lichen planus, lamellarichthyosis, epidermolytic hyperkeratosis, premalignant keratosis,pyoderma gangrenosum, allergic conditions and responses, allergicreaction, eczema including allergic or atopic eczema, asteatotic eczema,dyshidrotic eczema, and vesicular palmoplantar eczema, asthma such asasthma bronchiale, bronchial asthma, and auto-immune asthma, conditionsinvolving infiltration of T cells and chronic inflammatory responses,immune reactions against foreign antigens such as fetal A-B—O bloodgroups during pregnancy, chronic pulmonary inflammatory disease,autoimmune myocarditis, leukocyte adhesion deficiency, lupus, includinglupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus,extra-renal lupus, discoid lupus and discoid lupus erythematosus,alopecia lupus, systemic lupus erythematosus (SLE) such as cutaneous SLEor subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupuserythematosus disseminatus, juvenile onset (Type I) diabetes mellitus,including pediatric insulin-dependent diabetes mellitus (IDDM), adultonset diabetes mellitus (Type II diabetes), autoimmune diabetes,idiopathic diabetes insipidus, diabetic retinopathy, diabeticnephropathy, diabetic large-artery disorder, immune responses associatedwith acute and delayed hypersensitivity mediated by cytokines andT-lymphocytes, tuberculosis, sarcoidosis, granulomatosis includinglymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis,vasculitides, including vasculitis, large-vessel vasculitis (includingpolymyalgia rheumatica and giant-cell (Takayasu's) arteritis),medium-vessel vasculitis (including Kawasaki's disease and polyarteritisnodosa/periarteritis nodosa), microscopic polyarteritis,immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivityvasculitis, necrotizing vasculitis such as systemic necrotizingvasculitis, and ANCA-associated vasculitis, such as Churg-Straussvasculitis or syndrome (CSS) and ANCA-associated small-vesselvasculitis, temporal arteritis, aplastic anemia, autoimmune aplasticanemia, Coombs positive anemia, Diamond Blackfan anemia, hemolyticanemia or immune hemolytic anemia including autoimmune hemolytic anemia(AIHA), pernicious anemia (anemia pemiciosa), Addison's disease, purered cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A,autoimmune neutropenia, pancytopenia, leukopenia, diseases involvingleukocyte diapedesis, CNS inflammatory disorders, multiple organ injurysyndrome such as those secondary to septicemia, trauma or hemorrhage,antigen-antibody complex-mediated diseases, anti-glomerular basementmembrane disease, anti-phospholipid antibody syndrome, allergicneuritis, Behcet's disease/syndrome, Castleman's syndrome, Goodpasture'ssyndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnsonsyndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid,pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigusmucus-membrane pemphigoid, and pemphigus erythematosus), autoimmunepolyendocrinopathies, Reiter's disease or syndrome, thermal injury,preeclampsia, an immune complex disorder such as immune complexnephritis, antibody-mediated nephritis, polyneuropathies, chronicneuropathy such as IgM polyneuropathies or IgM-mediated neuropathy,thrombocytopenia (as developed by myocardial infarction patients, forexample), including thrombotic thrombocytopenic purpura (TTP),post-transfusion purpura (PTP), heparin-induced thrombocytopenia, andautoimmune or immune-mediated thrombocytopenia such as idiopathicthrombocytopenic purpura (ITP) including chronic or acute ITP, scleritissuch as idiopathic cerato-scleritis, episcleritis, autoimmune disease ofthe testis and ovary including autoimmune orchitis and oophoritis,primary hypothyroidism, hypoparathyroidism, autoimmune endocrinediseases including thyroiditis such as autoimmune thyroiditis,Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), orsubacute thyroiditis, autoimmune thyroid disease, idiopathichypothyroidism, Grave's disease, polyglandular syndromes such asautoimmune polyglandular syndromes (or polyglandular endocrinopathysyndromes), paraneoplastic syndromes, including neurologicparaneoplastic syndromes such as Lambert-Eaton myasthenic syndrome orEaton-Lambert syndrome, stiff-man or stiff-person syndrome,encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergica and experimental allergic encephalomyelitis(EAE), myasthenia gravis such as thymoma-associated myasthenia gravis,cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonusmyoclonus syndrome (OMS), and sensory neuropathy, multifocal motorneuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis,lupoid hepatitis, giant-cell hepatitis, chronic active hepatitis orautoimmune chronic active hepatitis, lymphoid interstitial pneumonitis(LIP), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barresyndrome, Berger's disease (IgA nephropathy), idiopathic IgAnephropathy, linear IgA dermatosis, acute febrile neutrophilicdermatosis, subcorneal pustular dermatosis, transient acantholyticdermatosis, cirrhosis such as primary biliary cirrhosis andpneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliacdisease, celiac sprue (gluten enteropathy), refractory sprue, idiopathicsprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; LouGehrig's disease), coronary artery disease, autoimmune ear disease suchas autoimmune inner ear disease (AIED), autoimmune hearing loss,polychondritis such as refractory or relapsed or relapsingpolychondritis, pulmonary alveolar proteinosis, Cogan'ssyndrome/nonsyphilitic interstitial keratitis, Bell's palsy, Sweet'sdisease/syndrome, rosacea autoimmune, zoster-associated pain,amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis,which includes monoclonal B cell lymphocytosis (e.g., benign monoclonalgammopathy and monoclonal gammopathy of undetermined significance,MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathiessuch as epilepsy, migraine, arrhythmia, muscular disorders, deafness,blindness, periodic paralysis, and channelopathies of the CNS, autism,inflammatory myopathy, focal or segmental or focal segmentalglomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis,chorioretinitis, autoimmune hepatological disorder, fibromyalgia,multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastricatrophy, presenile dementia, demyelinating diseases such as autoimmunedemyelinating diseases and chronic inflammatory demyelinatingpolyneuropathy, Dressler's syndrome, alopecia greata, alopecia totalis,CREST syndrome (calcinosis, Raynaud's phenomenon, esophagealdysmotility, sclerodactyl), and telangiectasia), male and femaleautoimmune infertility, e.g., due to anti-spermatozoan antibodies, mixedconnective tissue disease, Chagas' disease, rheumatic fever, recurrentabortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome,Cushing's syndrome, bird-fancier's lung, allergic granulomatousangiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitissuch as allergic alveolitis and fibrosing alveolitis, interstitial lungdisease, transfusion reaction, leprosy, malaria, parasitic diseases suchas leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis,aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue,endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonaryfibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathicpulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatumet diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, flariasis, cyclitis such as chroniccyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), orFuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus(HIV) infection, SCID, acquired immune deficiency syndrome (AIDS),echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis,parvovirus infection, rubella virus infection, post-vaccinationsyndromes, congenital rubella infection, Epstein-Barr virus infection,mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea,post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis,tabes dorsalis, chorioiditis, giant-cell polymyalgia, chronichypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemickeratoconjunctivitis, idiopathic nephritic syndrome, minimal changenephropathy, benign familial and ischemia-reperfusion injury, transplantorgan reperfusion, retinal autoimmunity, joint inflammation, bronchitis,chronic obstructive airway/pulmonary disease, silicosis, aphthae,aphthous stomatitis, arteriosclerotic disorders, aspermiogenese,autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren'scontracture, endophthalmia phacoanaphylactica, enteritis allergica,erythema nodosum leprosum, idiopathic facial paralysis, chronic fatiguesyndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearingloss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,leucopenia, mononucleosis infectiosa, traverse myelitis, primaryidiopathic myxedema, nephrosis, ophthalmia symphatica, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,non-malignant thymoma, vitiligo, toxic-shock syndrome, food poisoning,conditions involving infiltration of T cells, leukocyte-adhesiondeficiency, immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes, diseasesinvolving leukocyte diapedesis, multiple organ injury syndrome,antigen-antibody complex-mediated diseases, antiglomerular basementmembrane disease, allergic neuritis, autoimmune polyendocrinopathies,oophoritis, primary myxedema, autoimmune atrophic gastritis, sympatheticophthalmia, rheumatic diseases, mixed connective tissue disease,nephrotic syndrome, insulitis, polyendocrine failure, autoimmunepolyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism(AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolisisbullosa acquisita (EBA), hemochromatosis, myocarditis, nephroticsyndrome, primary sclerosing cholangitis, purulent or nonpurulentsinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, orsphenoid sinusitis, an eosinophil-related disorder such as eosinophilia,pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome,Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonaryeosinophilia, bronchopneumonic aspergillosis, aspergilloma, orgranulomas containing eosinophils, anaphylaxis, seronegativespondyloarthritides, polyendocrine autoimmune disease, sclerosingcholangitis, sclera, episclera, chronic mucocutaneous candidiasis,Bruton's syndrome, transient hypogammaglobulinemia of infancy,Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis,autoimmune disorders associated with collagen disease, rheumatism,neurological disease, lymphadenitis, reduction in blood pressureresponse, vascular dysfunction, tissue injury, cardiovascular ischemia,hyperalgesia, renal ischemia, cerebral ischemia, and diseaseaccompanying vascularization, allergic hypersensitivity disorders,glomerulonephritides, reperfusion injury, ischemic re-perfusiondisorder, reperfusion injury of myocardial or other tissues,lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses withacute inflammatory components, multiple organ failure, bullous diseases,renal cortical necrosis, acute purulent meningitis or other centralnervous system inflammatory disorders, ocular and orbital inflammatorydisorders, granulocyte transfusion-associated syndromes,cytokine-induced toxicity, narcolepsy, acute serious inflammation,chronic intractable inflammation, pyelitis, endarterial hyperplasia,peptic ulcer, valvulitis, and endometriosis.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

“Recombinant” when used with reference, e.g., to a cell, or nucleicacid, protein, or vector, indicates that the cell, nucleic acid, proteinor vector, has been modified by the introduction of a heterologousnucleic acid or protein or the alteration of a native nucleic acid orprotein, or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found within thenative (non-recombinant) form of the cell or express native genes thatare otherwise abnormally expressed, under expressed or not expressed atall.

The phrase “nucleic acid” or “polynucleotide sequence” refers to asingle or double-stranded polymer of deoxyribonucleotide orribonucleotide bases read from the 5′ to the 3′ end. Nucleic acids canalso include modified nucleotides that permit correct read through by apolymerase and do not alter expression of a polypeptide encoded by thatnucleic acid, including, for example, conservatively modified variants.

“Polypeptide,” “peptide” and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymers. Polypeptides of the invention includeconservatively modified variants. One of skill will recognize thatsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alter, add or delete a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention. The following eight groups eachcontain amino acids that are conservative substitutions for oneanother: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (33). The term“conservative substitution” also includes the use of a substituted aminoacid in place of an unsubstituted parent amino acid provided that such apolypeptide also displays the requisite binding activity.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that function in amanner similar to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. “Amino acid analog” refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, i.e., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs have modified R groups (e.g., norleucine) or modified peptidebackbones but retain the same basic chemical structure as a naturallyoccurring amino acid. “Amino acid mimetic” refers to a chemical compoundhaving a structure that is different from the general chemical structureof an amino acid but that functions in a manner similar to a naturallyoccurring amino acid.

Amino acids can be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission (see Table 1 below).Nucleotides, likewise, can be referred to by their commonly acceptedsingle-letter codes.

TABLE 1 SYMBOL 1 -Letter 3 -Letter AMINO ACID Y Tyr L-tyrosine G GlyL-glycine F Phe L-phenylalanine M Met L-methionine A Ala L-alanine S SerL-serine I Ile L-isoleucine L Leu L-leucine T Thr L-threonine V ValL-valine P Pro L-proline K Lys L-lysine H His L-histidine Q GlnL-glutamine E Glu L-glutamic acid W Trp L-tryptophan R Arg L-arginine DAsp L-aspartic acid N Asn L-asparagine C Cys L-cysteine

It should be noted that all amino acid sequences are represented hereinby formulae whose left to right orientation is in the conventionaldirection of amino-terminus to carboxy-terminus.

As used herein, the term “in vitro” or “ex vivo” refers to an artificialenvironment and to processes or reactions that occur within anartificial environment, for example, but not limited to, test tubes andcell cultures. The term “in vivo” refers to a natural environment (e.g.,an animal or a cell) and to processes or reactions that occur within anatural environment.

“Pharmaceutically acceptable,” “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a human without theproduction of undesirable physiological effects to a degree that wouldprohibit administration of the composition.

The term “pharmaceutically acceptable carrier” refers to reagents,excipients, cells, compounds, materials, compositions, and/or dosageforms which are, within the scope of sound medical judgment, suitablefor use in contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other complicationcommensurate with a reasonable benefit/risk ratio. As described ingreater detail herein, pharmaceutically acceptable carriers suitable foruse in the present invention include gases, liquids, and semi-solid andsolid materials.

Except when noted, “subject” or “patient” are used interchangeably andrefer to mammals such as human patients and non-human primates, as wellas experimental animals such as rabbits, dogs, cats, rats, mice, andother animals. Accordingly, “subject” or “patient” as used herein meansany mammalian patient or subject to which the compositions of theinvention can be administered. In some embodiments of the presentinvention, the patient will be suffering from an infectious orinflammatory disease, or an autoimmune disease. In some embodiments ofthe present invention, the patient will have been diagnosed with cancer.In an exemplary embodiment of the present invention, to identifycandidate patients for treatment according to the invention, acceptedscreening methods are employed to determine the status of an existingdisease or condition in a subject or risk factors associated with atargeted or suspected disease or condition. These screening methodsinclude, for example, examinations to determine whether a subject issuffering from an infectious disease, an inflammatory disease, cancer,or an autoimmune disease. These and other routine methods allow theclinician to select subjects in need of therapy.

“Treating” refers to any indicia of success in the treatment oramelioration of, for example, an infectious disease, an inflammatorydisease such as a GM-CSF-mediated inflammatory disease, cancer, or anautoimmune disease, including any objective or subjective parameter suchas abatement; remission; diminishing of symptoms or making the diseasecondition more tolerable to the patient; slowing in the rate ofdegeneration or decline; or making the final point of degeneration lessdebilitating. The treatment or amelioration of symptoms can be based onobjective or subjective parameters; including the results of anexamination. Accordingly, the term “treating” includes theadministration of the compounds or agents of the present invention todelay, to alleviate, or to arrest or inhibit development of the symptomsor conditions associated with cancer, an infectious disease, aninflammatory disease such as a GM-CSF-mediated inflammatory disease, oran autoimmune disease. Treating includes, for example, inhibition ofgrowth of dysplastic cells, inhibition of the progression of cancer orneoplastic disease, maintenance of inhibited tumor growth, and inductionof remission.

“Therapeutic compound” as used herein refers to a compound useful in theprophylaxis or treatment of a disease or condition such as cancer, aninfectious disease, an inflammatory disease, or an autoimmune disease.

“Therapeutic effect” refers to the reduction, elimination, or preventionof the disease, symptoms of the disease, or side effects of the diseasein the subject. “Effective amount” refers to an amount necessary toproduce a desired effect. A “therapeutically effective amount” means theamount that, when administered to a subject for treating a disease,condition or disorder, is sufficient to effect treatment for thatdisease.

“Concomitant administration,” “concurrent administration,” or“co-administration” as used herein includes administration of the activeagents (e.g., MAbs, chemotherapeutic agents, biomolecules), inconjunction or combination, together, or before or after each other. Themultiple agent(s) may be administered by the same or by differentroutes, simultaneously or sequentially, as long as they are given in amanner sufficient to allow all agents to achieve effectiveconcentrations at the site of action. A person of ordinary skill in theart would have no difficulty determining the appropriate timing,sequence, and dosages of administration for particular drugs andcompositions of the present invention.

“Donor cells” is used broadly to refer to cells fused to human B-cellsto generate hybridomas. The cells include but are not limited to rodentmyelomas as understood by those skilled in the art; rodent cell lines,human cell lines; avian cell lines. Cell lines may be derived by anymeans known by those skilled in the art.

“Immunoglobulin” or “antibody” is used broadly to refer to both antibodymolecules and a variety of antibody-derived molecules and includes anymember of a group of glycoproteins occurring in higher mammals that aremajor components of the immune system. The term “antibody” is used inthe broadest sense and specifically covers monoclonal antibodies,antibody compositions with polyepitopic specificity, bispecificantibodies, diabodies, and single-chain molecules, as well as antibodyfragments (e.g., Fab, F(ab′)2, and Fv), so long as they exhibit thedesired biological activity. An immunoglobulin molecule includes antigenbinding domains, which each include the light chains and theend-terminal portion of the heavy chain, and the Fc region, which isnecessary for a variety of functions, such as complement fixation. Thereare five classes of immunoglobulins wherein the primary structure of theheavy chain, in the Fc region, determines the immunoglobulin class.Specifically, the alpha, delta, epsilon, gamma, and mu chains correspondto IgA, IgD, IgE, IgG and IgM, respectively. As used herein“immunoglobulin” or “antibody” includes all subclasses of alpha, delta,epsilon, gamma, and mu and also refers to any natural (e.g., IgA andIgM) or synthetic multimers of the four-chain immunoglobulin structure.Antibodies non-covalently, specifically, and reversibly bind an antigen.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that can be present inminor amounts. For example, monoclonal antibodies may be produced by asingle clone of antibody-producing cells. Unlike polyclonal antibodies,monoclonal antibodies are monospecific (e.g., specific for a singleepitope of a single antigen). The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention can be made by the hybridoma method first described byKohler et al., Nature, 256: 495, 1975, or can be made by recombinant DNAmethods. The “monoclonal antibodies” can also be isolated from phageantibody libraries using the techniques described in Marks et al., J.Mol. Biol., 222: 581-597, 1991, for example.

As used herein “chimerized” refers to an immunoglobulin, wherein theheavy and light chain variable regions are not of human origin andwherein the constant regions of the heavy and light chains are of humanorigin.

“Humanized” refers to an immunoglobulin such as an antibody, wherein theamino acids directly involved in antigen binding, the complementaritydetermining regions (CDR), of the heavy and light chains are not ofhuman origin, while the rest of the immunoglobulin molecule, theframework regions of the variable heavy and light chains and theconstant regions of the heavy and light chains, are of human origin.

“Fully human” refers to an immunoglobulin, such as an antibody, wherethe whole molecule is of human origin or consists of an amino acidsequence identical to a human form of the antibody.

“Epitope” refers to an immunological determinant of an antigen thatserves as an antibody-binding site. As used herein, the term“conformational epitope” refers to a discontinuous epitope formed by aspatial relationship between amino acids of an antigen other than anunbroken series of amino acids.

“Hybridoma” refers to the product of a cell-fusion between a culturedneoplastic lymphocyte and a primed B- or T-lymphocyte which expressesthe specific immune potential of the parent cell.

“GM-CSF” refers to a family of glycoprotein growth factors that controlthe production, differentiation, and function of granulocytes andmonocytes-macrophages. Exemplary, but by no means the only form of suchmolecules, can be seen in U.S. Pat. No. 5,602,007 (34), incorporated byreference.

As used herein the term “biomolecule” refers to any molecule that can beconjugated to, coadministered with, administered before or afteradministering an antibody, or otherwise used in association with anantibody of the invention. Biomolecules include, but are not limited to,enzymes, proteins, peptides, amino acids, nucleic acids, lipids,carbohydrates, and fragments, homologs, analogs, or derivatives, andcombinations thereof. Examples of biomolecules include but are notlimited to interleukin-2, interferon alpha, interferon beta, interferongamma, rituxan, zevalin, herceptin, erbitux, and avastin. Thebiomolecules can be native, recombinant, or synthesized, and may bemodified from their native form with, for example, glycosylations,acetylations, phosphorylations, myristylations, and the like. The termbiomolecule as it is used herein is not limited to naturally occurringmolecules, and includes synthetic molecules having no biological origin.

Polypeptides in accordance with the present invention can be synthesizedfrom amino acids by techniques known to those skilled in the polypeptideart. In general, these methods comprise the sequential addition of oneor more amino acid residues or suitably protected amino acid residues toa growing peptide chain. Normally, either the amino or carboxyl group ofthe first amino acid residue is protected by a suitable, selectivelyremovable protecting group. A different, selectively removableprotecting group is utilized for amino acids containing a reactive sidegroup (e.g., lysine).

Various methods of preparing polypeptides of the present invention areknown in the art (WO 89/06657; WO 92/22315; WO 98/49191; U.S. Pat. Nos.5,260,273; 5,164,369; 5,407,914; 5,789,381; 5,952,303; 6,013,619;6,013,764; 6,120,795; 6,613,734).

Additional residues can be added at either terminus of a polypeptide ofthe present invention, such as for the purpose of providing a “linker”by which such a polypeptide can be conveniently affixed to a label orsolid matrix, or carrier. Labels, solid matrices and carriers that canbe used with the polypeptides of this invention are known in the art;some examples are also described herein.

Amino acid residue linkers are usually at least one residue and can be40 or more residues, more often 1 to 10 residues. Typical amino acidresidues used for linking are tyrosine, cysteine, lysine, glutamic andaspartic acid, or the like. In addition, a polypeptide sequence of thisinvention can differ from the natural sequence by the sequence beingmodified by terminal-NH₂ acylation, e.g., acetylation, or thioglycolicacid amidation, terminal-carboxylamidation, e.g., ammonia, methylamine,and the like.

While it is appreciated that many useful polypeptides disclosed herein,e.g., SEQ ID NOs: 1-5, it is also true that a wide variety of othermolecules, including uncommon but naturally occurring amino acids,metabolites and catabolites of natural amino acids, substituted aminoacids, and amino acid analogs, as well as amino acids in the “D”configuration, are useful in molecules and compositions of the presentinvention. In addition, “designed” amino acid derivatives, analogs andmimetics are also useful in various compounds, compositions and methodsof the present invention, as well as polymers including backbonestructures composed of non-amide linkages.

As used herein, “analogs” and “derivatives” of polypeptides and aminoacid residues are intended to encompass metabolites and catabolites ofamino acids, as well as molecules which include linkages, backbones,side-chains or side-groups which differ from those ordinarily found inwhat are termed “naturally-occurring” L-form amino acids. (The terms“analog” and “derivative” can also conveniently be used interchangeablyherein). Thus, D-amino acids, molecules which mimic amino acids andamino acids with “designed” side chains (i.e., that can substitute forone or more amino acids in a molecule having surfactant activity) arealso encompassed by the terms “analogs” and “derivatives” herein.

For example, in addition to the L-amino acids listed in Table 1, aminoacid metabolites such as homoarginine, citrulline, ornithine, andα-aminobutanoic acid are also useful in molecules and compositions ofthe present invention.

In another variation, one can wish to construct a molecule that adopts amore “rigid” conformation; one means of accomplishing this would be toadd methyl or other groups to the α carbon atom of the amino acids.

Further, substituted amino acids which are not generally derived fromproteins, but which are known in nature, are useful as disclosed herein,include the following examples: L-canavanine; 1-methyl-L-histidine;3-methyl-L-histidine; 2-methyl L-histidine; α,ε-diaminopimelic acid (Lform, meso form, or both); sarcosine; L-ornithine betaine; betaine ofhistidine (herzynine); L-citrulline; L-phosphoarginine; D-octopine;o-carbamyl-D-serine; γ-aminobutanoic acid; and β-lysine. D-amino acidsand D-amino acid analogs, including the following, are also useful inproteins, peptides and compositions of the present invention: D-alanine,D-serine, D-valine, D-leucine, D-isoleucine, D-alloisoleucine,D-phenylalanine, D-glutamic acid, D-proline, and D-allohydroxyproline,and the like. The foregoing can also be used in GM-CSF polypeptidesaccording to the present invention.

It should also be appreciated that the present invention encompasses awide variety of modified amino acids, including analogs, metabolites,catabolites, and derivatives, irrespective of the time or location atwhich modification occurs. In essence, one can place modified aminoacids into three categories: (1) catabolites and metabolites of aminoacids; (2) modified amino acids generated via posttranslationalmodification (e.g., modification of side chains); and (3) modificationsmade to amino acids via non-metabolic or non-catabolic processes (e.g.,the synthesis of modified amino acids or derivatives in the laboratory).

The present invention also contemplates that one can readily design sidechains of the amino acids of residue units that include longer orshortened side chains by adding or subtracting methylene groups ineither linear, branched chain, or hydrocarbon or heterocyclic ringarrangements. The linear and branched chain structures can also containnon-carbon atoms such as S, O, or N. Fatty acids can also be usefulconstituents of surfactant molecules herein. The designed side chainscan terminate with (R′) or without (R) charged or polar groupappendages.

Analogs, including molecules resulting from the use of differentlinkers, are also useful in the peptides of the invention. Moleculeswith side chains linked together via linkages other than the amidelinkage, e.g., molecules containing amino acid side chains or other sidechains (R- or R′-) wherein the components are linked via carboxy- orphospho-esters, ethylene, methylene, ketone or ether linkages, to name afew examples, are also useful as disclosed herein. In essence, any aminoacid side chain, R or R′ group-containing molecule can be useful asdisclosed herein.

The present invention also contemplates molecules comprising peptidedimers joined by an appropriate linker, e.g., peptide dimers linked bycysteine molecules. (As those of skill in the art are aware, twocysteine molecules can be linked together by a disulfide bridge formedby oxidation of their thiol groups). Such linkers or bridges can thuscross-link different polypeptide chains, dimers, trimers, and the like.Other useful linkers which can be used to connect peptide dimers and/orother peptide multimers include those listed above, e.g., carboxy- orphospho-ester, ethylene, methylene, ketone or ether linkages, and thelike.

One of skill in the art will appreciate that one can make a variety ofmodifications to individual amino acids, to the linkages, and/or to thechain itself, which modifications will produce molecules falling withinthe scope of the present invention, as long as the resulting moleculepossesses biological (e.g., antigenic) activity as described herein.

Preferred antigenic polypeptides of the invention are antigenic peptidesof GM-CSF (SEQ ID NO:1), and preferably of mature GM-CSF (SEQ ID NO:2).In some embodiments, the antigenic peptide comprises at least 5consecutive amino acids of the amino acid sequence shown in SEQ ID NO:1or SEQ ID NO:2. In other embodiments, the antigenic peptide comprises atleast 10 consecutive amino acids of the amino acid sequence shown in SEQID NO:1 or SEQ ID NO:2. In other embodiments, the antigenic peptidecomprises at least 15 consecutive amino acids of the amino acid sequenceshown in SEQ ID NO:1 or SEQ ID NO:2. In other embodiments, theimmunogenic portion comprises at least 20 consecutive amino acids of theamino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. In still otherembodiments, the immunogenic portion comprises at least 25 consecutiveamino acids of the amino acid sequence shown in SEQ ID NO:1 or SEQ IDNO:2. In certain preferred embodiments of the invention, the antigenicpeptide of the GM-CSF protein is amino acids 14-28 (SEQ ID NO:3), aminoacids 9-23 (SEQ ID NO:4), or amino acids 80-94 (SEQ ID NO:5) of theamino acid sequence of mature GM-CSF (SEQ ID NO:2).

In some embodiments of the invention, GM-CSF or antigenic peptidesthereof are conjugated to an immunogenic protein to enhance theimmunogenicity of the antigen. The immunogenic protein may be anyprotein that enhances the immune response of the cells, such as, but notlimited to tetanus toxoid C (TT), keyhole limpet hemocyanin (KLH),albumin, ovalbumin, chick albumin (CAB), bovine serum albumin,thyroglobulin, diptheria toxoid, BCG, cholera toxin and the like. Insome embodiments, the antigen is generated by denaturing the matureprotein.

Previously reported methods for generation of hybridomas secreting humanMAbs (WO2004/046330) using primary human B-cells were employed herein.Peripheral blood mononuclear cells, preferably human PBMCs, areimmunized ex vivo in the presence of target antigen and thenimmortalized via cell fusion with donor cells. Alternatively, selectedPBMCs are identified whose sera have high immune reactivity to antigenof interest.

Hybrid cells derived from the donor cells are screened for secretion oftarget antigen-specific MAbs. In some embodiments are provided methodsfor producing hybridoma cells producing monoclonal antibodies against atarget antigen from ex vivo immunized immunoglobulin-producing cellscomprising: (a) combining peripheral blood mononuclear cells comprisingimmunoglobulin-producing cells with a target antigen ex vivo; (b) fusingthe immunoglobulin-producing cells with donor cells to form hybridomacells; (c) determining binding of antigen by antibodies produced fromthe hybridoma cells; and (d) selecting hybridoma cells that produceantibodies that bind the target antigen; thereby generating hybridomacells that produce antibodies against the target antigen. In a preferredembodiment, the PBMCs are from healthy donors. The target antigen ispreferably GM-CSF and more preferably comprises an amino acid sequenceof one of SEQ ID NOs:35-38.

Alternatively, methods for generating hybridomas that produce monoclonalantibodies against target disease-associated antigen comprise the stepsof: (a) fusing ex vivo peripheral blood mononuclear cells comprisingimmunoglobulin-producing B-cells derived from a patient having a diseaseor an antigen-exposed donor with donor cells to form hybridoma cells;(b) determining binding by the antibodies produced from the hybridomacells to target antigen; and (c) selecting hybridoma cells that produceantibodies that bind to the target antigen; thereby producing hybridomacells that express antibodies against the target disease. Preferably,the target antigen is a disease-associated antigen, and preferably isassociated with cancer, an infectious disease, or an autoimmune disease.More preferably, the disease associated antigen is GM-CSF. The targetantigen is preferably GM-CSF and more preferably comprises an amino acidsequence of one of SEQ ID NOs:35-38. In a preferred embodiment, theantigen-exposed donor has been exposed to GM-CSF or may have pulmonaryalveolar proteinosis (PAP).

The invention provides methods for producing hybridoma cells producingantibodies to target antigen (e.g., GM-CSF or antigenic peptidesthereof) from ex vivo-immunized immunoglobulin-producing cellscomprising: (a) combining peripheral blood mononuclear cells comprisingimmunoglobulin-producing cells with target antigen ex vivo; (b) fusingthe immunoglobulin-producing cells with donor cells to form hybridomacells; (c) performing a screen for binding of immunoglobulins producedby the hybridoma cells to target antigen; thereby producing hybridomacells producing antibodies to target antigen. In a preferred embodiment,the PBMCs are from healthy donors. In some embodiments, the targetantigen comprises an antigenic polypeptide of the invention. The targetantigen preferably comprises an amino acid sequence of one of SEQ IDNOs: 1-5, preferably any one of SEQ ID NOs:3-5.

Alternatively, the invention provides methods for producing hybridomacells producing antibodies to a target antigen (e.g., GM-CSF, orantigenic peptides thereof) comprising: (a) selecting peripheral bloodmononuclear cells comprising immunoglobulin-producing cells from anantigen-exposed donor; (b) fusing the immunoglobulin-producing cellswith myeloma cells to form hybridoma cells; (c) performing a screen forbinding of immunoglobulins produced by the hybridoma cells to a targetantigen; thereby producing hybridoma cells producing antibodies to thetarget antigen. In some embodiments, the target antigen comprises anantigenic polypeptide of the invention. The target antigen preferablycomprises an amino acid sequence of one of SEQ ID NOs:1-5, morepreferably SEQ ID NOs:3-5. In a preferred embodiment, the donor has beenexposed to GM-CSF, or antigenic peptides thereof, or may have pulmonaryalveolar proteinosis (PAP).

In some embodiments, the donor (e.g., myeloma) cells express a proteininhibitor of mismatch repair. In some aspects, the hybridoma cellsexpress a protein inhibitor of mismatch repair. In some embodiments ofthe method of the invention, the protein inhibitor of mismatch repair isintroduced into the hybridoma cell after the fusion of the myeloma withthe immunoglobulin-producing cells. In other embodiments, the proteininhibitor of mismatch repair is introduced into the donor or myelomacell prior to the fusion with the immunoglobulin-producing cells. Instill other embodiments, the donor or myeloma cells or antibodyproducing cells are naturally deficient in mismatch repair.

Protein inhibitors of mismatch repair include dominant negative allelesof mismatch repair genes. Dominant negative alleles of mismatch repairgenes include but are not limited to dominant negative alleles of PMS2,PMS1, PMSR3, PMSR2, PMSR6, MLH1, GTBP, MSH3, MSH2, MLH3, or MSH1, andhomologs of mutL and mutS genes. In addition, polypeptides capable ofinterfering in mismatch repair may be used. For example, a dominantnegative allele of mutL PMS2 comprises the first 133 amino acids PMS2.Further delineation of amino acids in mutL homologs reveals amino acidsLSTAVKELVENSLDAGATNIDLKLKDYGVDLIEVSDNGCGVEEENFE (SEQ ID NO:6) andLRQVLSNLLDNAIKYTPEGGEITVSLERDGDHLEITVEDNGPGIPEEDLE (SEQ ID NO:7) orfragments thereof. Protein inhibitors of mismatch repair thus includepolypeptides of SEQ ID NOs: 6 and 7 and fragments thereof. In preferredembodiments, the protein inhibitor of mismatch repair is inactivated.For example, the protein inhibitor of mismatch repair may be inactivatedbefore or after identification of a hybridoma cell that generatesmonoclonal antibodies to the target antigen. Inactivation of the proteininhibitor of mismatch repair may be by any means known in the art, forexample, removal of an inducer or removal of the protein inhibitor ofmismatch repair from the cell (i.e., curing the cell of the proteininhibitor of mismatch repair). Inactivation of the inhibitor of mismatchrepair stabilizes the genome of the hypermutated hybridoma.

In some embodiments of the methods of generating hybridoma cells of theinvention, the hybridoma cells are exposed to a chemical inhibitor ofmismatch repair. Chemical inhibitors of mismatch repair used in certainembodiments of the methods of the invention include, but are not limitedto, at least one of an anthracene, an ATPase inhibitor, a nucleaseinhibitor, an RNA interference molecule, a polymerase inhibitor and anantisense oligonucleotide that specifically hybridizes to a nucleotideencoding a mismatch repair protein (WO2004/046330). In preferredembodiments, the chemical inhibitor is an anthracene compound having theformula:

wherein R₁-R₁₀ are independently hydrogen, hydroxyl, amino, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,O-alkynyl, S-alkynyl, N-alkynyl, aryl, substituted aryl, aryloxy,substituted aryloxy, heteroaryl, substituted heteroaryl, aralkyloxy,arylalkyl, alkylaryl, alkylaryloxy, arylsulfonyl, alkylsulfonyl,alkoxycarbonyl, aryloxycarbonyl, guanidino, carboxy, an alcohol, anamino acid, sulfonate, alkyl sulfonate, CN, NO₂, an aldehyde group, anester, an ether, a crown ether, a ketone, an organosulfur compound, anorganometallic group, a carboxylic acid, an organosilicon or acarbohydrate that optionally contains one or more alkylated hydroxylgroups; wherein said heteroalkyl, heteroaryl, and substituted heteroarylcontain at least one heteroatom that is oxygen, sulfur, a metal atom,phosphorus, silicon or nitrogen; and wherein said substituents of saidsubstituted alkyl, substituted alkenyl, substituted alkynyl, substitutedaryl, and substituted heteroaryl are halogen, CN, NO₂, lower alkyl,aryl, heteroaryl, aralkyl, aralkoxy, guanidino, alkoxycarbonyl, alkoxy,hydroxy, carboxy and amino; and wherein said amino groups are optionallysubstituted with an acyl group, or 1 to 3 aryl or lower alkyl groups. Incertain embodiments, R₅ and R₆ are hydrogen. In other embodiments,R₁-R₁₀ are independently hydrogen, hydroxyl, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, phenyl, tolyl, hydroxymethyl, hydroxypropyl,or hydroxybutyl. Non-limiting examples of the anthracenes include1,2-dimethylanthracene, 9,10-dimethylanthracene, 7,8-dimethylanthracene,9,10-duphenylanthracene, 9,10-dihydroxymethylanthracene,9-hydroxymethyl-10-methylanthracene, dimethylanthracene-1,2-diol,9-hydroxymethyl-10-methylanthracene-1,2-diol,9-hydroxymethyl-10-methylanthracene-3,4-diol, and9,10-di-m-tolylanthracene.

The chemical inhibitor may be introduced into the growth medium of thecells. In some embodiments, the chemical inhibitor may be withdrawn fromthe hypermutated hybridoma cells in order to re-stabilize the genome ofthe cells. Alternatively, the method may comprise inactivation of thechemical inhibitor of mismatch repair, thereby stabilizing the genome ofthe hypermutated hybridoma.

In some embodiments, the methods further comprise cloning theimmunoglobulin-producing genes from the antibody-producing cells andtransfecting the immunoglobulin genes into a mammalian expression cell,wherein the immunoglobulin genes are operably linked to expressioncontrol sequences.

The invention also provides methods for producing mammalian expressioncells that produce high affinity antibodies to target antigen from exvivo immunized immunoglobulin-producing cells comprising: (a) combiningperipheral blood mononuclear cells comprising immunoglobulin-producingcells with a target antigen ex vivo; (b) fusing theimmunoglobulin-producing cells with donor cells to form hybridoma cells;(c) determining binding of antigen by antibodies produced from thehybridoma cells; (d) optimizing antibody production of the hybridoma byinhibiting mismatch repair; or cloning immunoglobulin genes from thehybridoma into a mammalian expression cell, wherein the mismatch repairof the mammalian expression cell is inhibited; and (e) selectinghybridoma cells that produce antibodies that bind the target antigen;thereby generating hybridoma cells that produce antibodies against thetarget antigen. As an alternative to steps (a) and (b), fusion ex vivoof peripheral blood mononuclear cells comprisingimmunoglobulin-producing B-cells derived from a patient having a diseaseor an antigen-exposed donor with donor cells to form hybridoma cells maybe used.

The invention also provides methods for producing mammalian expressioncells that produce high affinity antibodies to target antigen from exvivo immunized immunoglobulin-producing cells comprising: (a) combiningperipheral blood mononuclear cells comprising immunoglobulin-producingcells with target antigen ex vivo; (b) fusing theimmunoglobulin-producing cells with myeloma cells to form hybridomacells; (c) performing a screen for binding of antibodies produced fromsaid hybridoma cells to target antigen; (d) optimizing production of thehybridoma by inhibiting mismatch repair; or cloning immunoglobulin genesfrom the hybridoma into a mammalian expression cell, wherein themismatch repair of the mammalian expression cell is inhibited; and (e)performing a screen for mammalian expression cells that secreteantibodies with higher affinity for target antigen as compared toantibodies produced from the hybridoma or recombinant cells.

The invention also provides a method for producing mammalian expressioncells that produce high titers of high-affinity antibodies from ex vivoimmunized immunoglobulin-producing cells comprising: (a) combiningperipheral blood mononuclear cells comprising immunoglobulin-producingcells with a target antigen ex vivo; (b) fusing theimmunoglobulin-producing cells with donor cells to form hybridoma cells;(c) determining binding of antigen by antibodies produced from thehybridoma cells; (d) cloning immunoglobulin genes from the hybridomainto a parental mammalian expression cell, wherein mismatch repair ofthe mammalian expression cell is inhibited; (e) incubating the parentalmammalian cell or hybridoma expression cell to allow for mutagenesis,thereby forming hypermutated mammalian expression cells; (f) selectinghypermutable mammalian expression cells that secrete antibodies withhigher affinity for target antigen as compared to antibodies producedfrom the parental hybridoma cells or selecting hypermutable mammalianexpression cells that secrete higher titers of antibodies than parentalmammalian expression cells; thereby producing mammalian expression cellsthat produce antibodies to target antigen from ex vivo immunizedimmunoglobulin-producing cells. As an alternative to steps (a) and (b),fusion ex vivo of peripheral blood mononuclear cells comprisingimmunoglobulin-producing B-cells derived from a patient having a diseaseor an antigen-exposed donor with donor cells to form hybridoma cells maybe used.

The invention also provides a method for producing mammalian expressioncells that produce high titers of high-affinity antibodies from ex vivoimmunized immunoglobulin-producing cells comprising: (a) combiningperipheral blood mononuclear cells comprising immunoglobulin-producingcells with target antigen ex vivo; (b) fusing theimmunoglobulin-producing cells with myeloma cells to form hybridomacells; (c) performing a screen for binding of antibodies produced fromthe hybridoma cells to antigen; (d) cloning immunoglobulin genes fromthe hybridoma into a parental mammalian expression cell, whereinmismatch repair of the mammalian expression cell is inhibited oroptimizing production of the hybridoma by inhibiting mismatch repair;(e) incubating the parental mammalian cell or hybridoma expression cellto allow for mutagenesis, thereby forming hypermutated mammalianexpression cells; (f) performing a screen of hypermutable mammalianexpression cells that secrete antibodies with higher affinity for targetantigen as compared to antibodies produced from the hybridoma cells; and(g) performing a screen of hypermutable mammalian expression cells thatsecrete higher titers of antibodies than parental mammalian expressioncells; thereby producing mammalian expression cells that produce hightiters of high-affinity antibodies from ex vivo immunizedimmunoglobulin-producing cells.

In some embodiments of the method of the invention, antibodies arescreened using an ELISA-based assay or other assays that can measureantibody-antigen binding known in the art. Crowther, J. R. (2001) TheELISA guidebook, 1st ed. Humana Press, Totowa, N.J.

In some embodiments, the screening assays screen for hypermutatedhybridomas that produce higher affinity antibodies than those producedby the parental hybridomas.

In some embodiments, the method of the invention further comprisesselecting hypermutated antibody-producing cells having higher titers ofantibodies than that produced by the originally selected cells.

Methods of fusion of immunoglobulin-producing cells to myeloma cells andmyeloma cells useful in such methods also are known in the art. Kohler &Milstein, Eur. J. Immunol. 1976. 6:511-9. Derivation of specificantibody-producing tissue culture and tumor lines by cell fusion.

Human B-cells used for the generation of MAbs designed foradministration to humans may represent a potential vehicle of viraltransmission. Fusion partner cells and peripheral blood mononuclearcells (PBMCs) from donors may be pre-screened to confirm absence ofviral DNA, for example, by PCR, including immunodeficiency-1 and 2,hepatitis B and C, cytomegalo-, herpes-6, and Epstein Barr viruses.

Hybridoma cells produced according to the methods of the invention areincluded within the scope of the invention.

The invention also comprises antibodies to target antigens produced bythe hybridoma cells generated according to the methods of the invention.Antibodies of the invention also comprise antibodies producedrecombinantly using the polynucleotides of the invention. Preferredantibodies of the invention are monoclonal antibodies. Antibodies of theinvention preferably are fully human, more preferably fully humanmonoclonal antibodies.

Preferred antibodies of the invention specifically bind an epitope, forexample a conformational epitope, of a target antigen. The antibodies ofthe invention preferably are directed against disease-associatedantigen, for example, but not limited to GM-CSF, preferably human GM-CSF(SEQ ID NO:1), more preferably mature human GM-CSF (SEQ ID NO:2). Insome embodiments, the epitope to which the antibody binds comprises atleast 5 consecutive amino acids of the amino acid sequence shown in SEQID NO:1 or SEQ ID NO:2. In other embodiments, the epitope to which theantibody binds comprises at least 10 consecutive amino acids of theamino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. In otherembodiments, the epitope to which the antibody binds comprises at least15 consecutive amino acids of the amino acid sequence shown in SEQ IDNO:1 or SEQ ID NO:2. In other embodiments, the epitope to which theantibody binds comprises at least 20 consecutive amino acids of theamino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. In still otherembodiments, the epitope to which the antibody binds comprises at least25 consecutive amino acids of the amino acid sequence shown in SEQ IDNO:1 or SEQ ID NO:2. In certain preferred embodiments of the invention,the epitope of GM-CSF to which the antibody binds comprises an aminoacid sequence of at least one of SEQ ID NOs:33-36. Antibody-producingcells have been placed with the Amer. Type Cult. Coll. (10801 UniversityBlvd., Manassas, Va. 20110-2209) on Jan. 18, 2007 (10G9), and on Feb. 2,2007 (E10) and have been assigned Access. Nos. PTA-8173 and PTA-8193.respectively. Examples of anti-GM-CSF antibodies of the invention areantibodies produced by such cells.

Those of skill in the art will recognize that antibody specificity isprimarily determined by the six CDR regions, especially H chain CDR3(Kala M et al. (2002) J. Biochem. 132:535-41; Morea V et al. (1998) J.Mol. Biol. 275:269-94; and, Chothia C et al. (1987) J. Mol. Biol.196:901-17). Antibody framework regions, however, can play a role inantigen-antibody interactions (Panka D J et al. (1988) Proc. Natl. Acad.Sci. USA 85:3080-4), particularly with respect to their role inconformation of CDR loops (Foote J et al. (1992) J. Mol. Biol.224:487-99). Thus, the inventive antibodies can comprise any combinationof H or L chain CDR or FWR regions that confer antibody specificity forGM-CSF. Domain shuffling experiments, which are routinely carried out inthe art (Jirholt P et al. (1998) Gene 215:471-6; Soderlind E et al.(2000) Nature Biotechnology 18:852-6), can be employed to generateantibodies that specifically bind GM-CSF according to the specificationsdescribed and exemplified herein. Antibodies generated by such domainshuffling experiments are within the scope of the present invention.

Accordingly, in some embodiments, the antibodies comprise a heavy chainCDR1 amino acid sequence substantially the same as or identical to SEQID NO:40 or 46. In some embodiments, the antibodies comprise a heavychain CDR2 amino acid sequence substantially the same as or identical toSEQ ID NO:41 or 47. In some particularly preferred embodiments, theantibodies comprise a heavy chain CDR3 amino acid sequence substantiallythe same as or identical to SEQ ID NO:42 or 48. In some embodiments, theantibodies comprise a light chain CDR1 amino acid sequence substantiallythe same as or identical to SEQ ID NO:43 or 49. In some embodiments, theantibodies comprise a light chain CDR2 amino acid sequence substantiallythe same as or identical to SEQ ID NO:44 or 50. In some embodiments, theantibodies comprise a light chain CDR3 amino acid sequence substantiallythe same as or identical to SEQ ID NO:45 or 51. In some embodiments, theantibodies comprise a heavy chain FWR1 amino acid sequence substantiallythe same as or identical to SEQ ID NO:52 or 58. In some embodiments, theantibodies comprise a heavy chain FWR2 amino acid sequence substantiallythe same as or identical to SEQ ID NO:53 or 59. In some embodiments, theantibodies comprise a heavy chain FWR3 amino acid sequence substantiallythe same as or identical to SEQ ID NO:54 or 60. In some embodiments, theantibodies comprise a light chain FWR1 amino acid sequence substantiallythe same as or identical to SEQ ID NO:55 or 61. In some embodiments, theantibodies comprise a light chain FWR2 amino acid sequence substantiallythe same as or identical to SEQ ID NO:56 or 62. In some embodiments, theantibodies comprise a light chain FWR3 amino acid sequence substantiallythe same as or identical to SEQ ID NO:57 or 63.

In some preferred embodiments, the antibody of the invention comprises aheavy chain comprising an amino acid sequence of SEQ ID NO:8, 9, or 16.The heavy chain can be encoded by a nucleic acid sequence comprising anucleotide sequence of SEQ ID NO:10, 11, or 17. In some preferredembodiments, the antibody of the invention comprises a light chaincomprising an amino acid sequence of SEQ ID NO:12, 13, or 18. The lightchain can be encoded by a nucleic acid sequence comprising a nucleotidesequence of SEQ ID NO:14, 15, or 19.

It is to be understood that because of the natural sequence variationlikely to exist among heavy and light chains and the genes encodingthem, one skilled in the art would expect to find some level ofvariation within the amino acid sequences or the genes encoding them,while still maintaining the unique binding properties (e.g., specificityand affinity) of the antibodies of the present invention. Such anexpectation is due in part to the degeneracy of the genetic code, aswell as to the known evolutionary success of conservative amino acidsequence variations, which do not appreciably alter the nature of theencoded protein. Accordingly, such variants and homologs are consideredsubstantially the same as one another and are included within the scopeof the present invention.

The antibodies of the invention thus include variants having single ormultiple amino acid substitutions, deletions, additions, or replacementsthat retain the biological properties (e.g., binding affinity or immuneeffector activity) of the antibodies of the invention. The skilledperson can produce variants having single or multiple amino acidsubstitutions, deletions, additions or replacements. These variants mayinclude, inter alia: (a) variants in which one or more amino acidresidues are substituted with conservative or nonconservative aminoacids, (b) variants in which one or more amino acids are added to ordeleted from the polypeptide, (c) variants in which one or more aminoacids include a substituent group, and (d) variants in which thepolypeptide is fused with another peptide or polypeptide such as afusion partner, a protein tag or other chemical moiety, that may conferuseful properties to the polypeptide, such as, for example, an epitopefor an antibody, a polyhistidine sequence, a biotin moiety and the like.Antibodies of the invention may include variants in which amino acidresidues from one species are substituted for the corresponding residuein another species, either at the conserved or nonconserved positions.In other embodiments, amino acid residues at nonconserved positions aresubstituted with conservative or nonconservative residues. Thetechniques for obtaining these variants, including genetic(suppressions, deletions, mutations, etc.), chemical, and enzymatictechniques, are known to the person having ordinary skill in the art.

In some preferred embodiments, the antibodies can comprise a heavy chainthat comprises the amino acid sequence of SEQ ID NO: 8 and a light chainthat comprises the amino acid sequence of SEQ ID NO:12. In somepreferred embodiments, the antibodies can comprise a heavy chain thatcomprises the amino acid sequence of SEQ ID NO:16 and a light chain thatcomprises the amino acid sequence of SEQ ID NO:18. In some embodiments,the antibodies can comprise a heavy chain that comprises the amino acidsequence of SEQ ID NO:9 and a light chain that comprises the amino acidsequence of SEQ ID NO:13. Those of skill in the art will recognize,however, that in some cases, the pairing of a given heavy with variouslight chains, or the pairing of a given light chain with various heavychains will produce antibodies with the same or better specificityand/or affinity than the native combination. Accordingly, the inventionis not limited to the preferred combinations of H and L chain pairs, andthe inventive antibodies thus encompass different combinations of H andL chain pairs, including without limitation, the H and L chainsdescribed herein, or other H or L chains that would be known to those ofskill in the art, or otherwise experimentally determined to becompatible with the H and L chains described herein in order to obtainspecific and high affinity binding to GM-CSF.

Preferred antibodies of the invention comprise two heavy chains.Preferred antibodies of the invention comprise two light chains. Morepreferred are antibodies comprising two heavy chains and two lightchains of the invention.

The antibodies of the invention include variants having single ormultiple amino acid substitutions, deletions, additions, or replacementsthat retain the biological properties (e.g., binding affinity or immuneeffector activity) of the antibodies of the invention. The skilledperson can produce variants having single or multiple amino acidsubstitutions, deletions, additions or replacements. These variants mayinclude, inter alia: (a) variants in which one or more amino acidresidues are substituted with conservative or nonconservative aminoacids, (b) variants in which one or more amino acids are added to ordeleted from the polypeptide, (c) variants in which one or more aminoacids include a substituent group, and (d) variants in which thepolypeptide is fused with another peptide or polypeptide such as afusion partner, a protein tag or other chemical moiety, that may conferuseful properties to the polypeptide, such as, for example, an epitopefor an antibody, a polyhistidine sequence, a biotin moiety and the like.Antibodies of the invention may include variants in which amino acidresidues from one species are substituted for the corresponding residuein another species, either at the conserved or nonconserved positions.In another embodiment, amino acid residues at nonconserved positions aresubstituted with conservative or nonconservative residues. Thetechniques for obtaining these variants, including genetic(suppressions, deletions, mutations, etc.), chemical, and enzymatictechniques, are known to the person having ordinary skill in the art.Antibodies of the invention also include antibody fragments. A“fragment” refers to polypeptide sequences which are preferably at leastabout 40, more preferably at least to about 50, more preferably at leastabout 60, more preferably at least about 70, more preferably at leastabout 80, more preferably at least about 90, and more preferably atleast about 100 amino acids in length, and which retain some biologicalactivity or immunological activity of the full-length sequence, forexample, binding affinity or avidity and immune effector activity.

The antibodies of the invention have binding affinities for targetantigen that include a dissociation constant (K_(D)) of less than1×10⁻². In some embodiments, the K_(D) is less than 1×10⁻³. In otherembodiments, the K_(D) is less than 1×10⁻⁴. In some embodiments, theK_(D) is less than 1×10⁻⁵. In still other embodiments, the K_(D) is lessthan 1×10⁻⁶. In other embodiments, the K_(D) is less than 1×10⁻⁷. Inother embodiments, the K_(D) is less than 1×10⁻⁸. In other embodiments,the K_(D) is less than 1×10⁻⁹. In other embodiments, the K_(D) is lessthan 1×10⁻¹⁰. In still other embodiments, the K_(D) is less than1×10⁻¹¹. In some embodiments, the K_(D) is less than 1×10⁻¹². In otherembodiments, the K_(D) is less than 1×10⁻¹³. In other embodiments, theK_(D) is less than 1×10⁻¹⁴. In still other embodiments, the K_(D) isless than 1×10⁻¹⁵.

Antibodies of the invention include derivatives that are modified, e.g.,by the covalent attachment of any type of molecule to the antibody suchthat covalent attachment does not prevent the antibody from binding toits epitope. Examples of suitable derivatives include, but are notlimited to glycosylated antibodies and fragments, acetylated antibodiesand fragments, pegylated antibodies and fragments, phosphorylatedantibodies and fragments, and amidated antibodies and fragments. Theantibodies of the invention may themselves be derivatized by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other proteins, and the like. Further, the antibodies of theinvention may contain one or more non-classical amino acids as describedabove. In some embodiments of the invention, GM-CSF or epitopes thereofare conjugated to an immunogenic protein to enhance the immunogenicityof the antigen. The immunogenic protein may be any protein that enhancesthe immune response of the cells, such as, but not limited to tetanustoxoid C (TT), keyhole limpet hemocyanin (KLH), albumin, ovalbumin,chick albumin (CAB), bovine serum albumin, thyroglobulin, diptheriatoxoid, BCG, cholera toxin and the like. In some embodiments, theantigen is generated by denaturing the mature protein.

The antibodies of the invention may have post-translational moietiesthat improve upon antibody activity or stability. These moieties includesulfur, methyl, carbohydrate, phosphorus as well as other chemicalgroups commonly found on immunoglobulin molecules.

Antibodies of the invention may be of any isotype. Whereby isotype ofantibody can be changes using in vivo class switching or by geneticengineering.

Nucleotide sequences that encode polypeptides of the invention areprovided. Nucleic acids of the invention include but are not limited togenomic DNA, DNA, cDNA, RNA, double- and single-stranded nucleic acids,and complementary sequences thereof.

Preferred polynucleotides of the invention include nucleic acidsequences encoding an amino acid sequence of SEQ ID NO:8 and/or SEQ IDNO:12. In some embodiments, the heavy chain of the antibody is encodedby a polynucleotide comprising SEQ ID NO:10. In some embodiments, thelight chain of the antibody is encoded by a polynucleotide comprisingSEQ ID NO:14. Polynucleotides comprising a nucleic acid sequenceencoding an amino acid sequence of SEQ ID NO:8 and an amino acidsequence of SEQ ID NO:12 also are provided. Preferably such apolynucleotide comprises a nucleic acid sequence of SEQ ID NO:10 and/orSEQ ID NO:14.

Preferred polynucleotides of the invention include nucleic acidsequences encoding an amino acid sequence of SEQ ID NO:16 and/or SEQ IDNO:18. In some embodiments, the heavy chain of the antibody is encodedby a polynucleotide comprising SEQ ID NO:17. In some embodiments, thelight chain of the antibody is encoded by a polynucleotide comprisingSEQ ID NO:19. Polynucleotides comprising a nucleic acid sequenceencoding an amino acid sequence of SEQ ID NO:16 and an amino acidsequence of SEQ ID NO:18 also are provided. Preferably such apolynucleotide comprises a nucleic acid sequence of SEQ ID NO:17 and/orSEQ ID NO:19.

In some embodiments, polynucleotides of the invention (and the peptidesthey encode) include a leader sequence. Any leader sequence known in theart may be employed. The leader sequence may include but is not limitedto a restriction site and/or a translation start site. For example, theinvention provides nucleic acid sequences encoding an amino acidsequence of SEQ ID NO:9 and/or SEQ ID NO:13. In some embodiments, theheavy chain of the antibody is encoded by a polynucleotide comprisingSEQ ID NO:11. In some embodiments, the light chain of the antibody isencoded by a polynucleotide comprising SEQ ID NO:15. Polynucleotidescomprising a nucleic acid sequence encoding an amino acid sequence ofSEQ ID NO:9 and an amino acid sequence of SEQ ID NO:13 also areprovided. Preferably such a polynucleotide comprises a nucleic acidsequence of SEQ ID NO:11 and/or SEQ ID NO:15.

Also contemplated by the invention are expression vectors comprising thepolynucleotides of the invention and host cells, such as but not limitedto recombinant host cells, expressing the polynucleotides of theinvention.

Recombinant expression vectors containing a sequence encoding apolypeptide of interest are provided. The expression vector may containone or more additional sequences such as but not limited to regulatorysequences (e.g., promoter, enhancer), a selection marker, and apolyadenylation signal.

Recombinant expression vectors of the invention include synthetic,genomic, or cDNA-derived nucleic acid fragments that encode at least onerecombinant protein which may be operably linked to suitable regulatoryelements. Such regulatory elements may include a transcriptionalpromoter, sequences encoding suitable mRNA ribosomal binding sites, andsequences that control the termination of transcription and translation.Expression vectors, especially mammalian expression vectors, may alsoinclude one or more nontranscribed elements such as an origin ofreplication, a suitable promoter and enhancer linked to the gene to beexpressed, other 5′ or 3′ flanking nontranscribed sequences, 5′ or 3′nontranslated sequences (such as necessary ribosome binding sites), apolyadenylation site, splice donor and acceptor sites, ortranscriptional termination sequences. An origin of replication thatconfers the ability to replicate in a host may also be incorporated.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells may be provided byviral sources. Exemplary vectors can be constructed as described inOkayama and Berg (1983) Mol. Cell. Biol. 3:280.

Selection markers that can be used in the system include those known inthe art, such as positive and negative selection markers, such as butnot limited to antibiotic resistance genes (e.g., neomycin resistancegene, a hygromycin resistance gene, a kanamycin resistance gene, atetracycline resistance gene, a penicillin resistance gene), HSV-TK,HSV-TK derivatives for ganciclovir selection, or bacterial purinenucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al.(2000) Gene Ther. 7:1738-1743). [0034] A nucleic acid sequence encodinga selection marker or the cloning site therefor may be upstream ordownstream of a nucleic acid sequence encoding a polypeptide of interestor cloning site therefor.

In some embodiments, the vector includes one or more promoters, such asbut not limited to a constitutive, inducible, host-specific, and/ortissue-specific promoter. For example, commonly used promoters andenhancers are derived from human cytomegalovirus (CMV), Adenovirus 2,Simian Virus 40 (SV40), and Polyoma. Viral genomic promoters, controland/or signal sequences may be utilized to drive expression which aredependent upon compatible host cells. Promoters derived fromhouse-keeping genes can also be used (e.g., the 3-globin, thymidinekinase, and the EF-1α promoters), depending on the identity of the celltype in which the vector is to be expressed. In some embodiments, apromoter is upstream of a nucleic acid sequence encoding one or morepolypeptides of interest.

Vectors of the invention may contain one or more Internal Ribosome EntrySite(s) (IRES). Inclusion of an IRES sequence into fusion vectors may bebeneficial for enhancing expression of some proteins.

In some embodiments the vector system will include one or morepolyadenylation sites (e.g., SV40), which may be upstream or downstreamof any of the aforementioned nucleic acid sequences.

Vector components may be contiguously linked, or arranged in a mannerthat provides optimal spacing for expressing the gene products (i.e., bythe introduction of “spacer” nucleotides between the ORFs), orpositioned in another way. Regulatory elements, such as the IRES motif,can also be arranged to provide optimal spacing for expression.

Cells transfected with expression vectors of the invention can beselected under positive selection conditions and/or screened forrecombinant protein expression. Recombinant-positive cells are expandedand screened for subclones exhibiting a desired phenotype.

Cells, including eukaryotic and prokaryotic cells, can be transformedwith the expression vectors of the invention. Accordingly, anotherembodiment of the invention provides a host cell transformed with anexpression vector of the instant invention. Cells of the invention arepreferably eukaryotic cells, more preferably cells of plant, rodent, orhuman origin, for example but not limited to NS0, CHO, perC.6, Tk-ts13,BHK, HEK293 cells, COS-7, T98G, CV-1/EBNA, L cells, C127, 3T3, HeLa,NS1, Sp2/0 myeloma cells, and BHK cell lines, among others.

In general, transfection will be carried out using a suspension ofcells, or a single cell, although other methods can also be applied tothe extent that sufficient fraction of the treated cells or tissueincorporates the polynucleotide, thereby allowing transfected cells tobe grown and utilized. Techniques for transfection are well known.Several transformation protocols are known in the art. See, e.g.,Kaufman (1988) Meth. Enzymology 185:537. As is readily understood bythose skilled in the art, the appropriate transformation protocol isdetermined by the host cell type and the nature of the gene of interest.The basic components of any such protocol include introducing nucleicacid sequence encoding the protein of interest into a suitable hostcell, and then identifying and isolating host cells which haveincorporated the vector DNA in a stable, expressible manner. Techniquesfor introducing polynucleotides include but are not limited toelectroporation, transduction, cell fusion, the use of calcium chloride,and packaging of the polynucleotide together with lipid for fusion withthe cells of interest. If the transfection is stable, such that theselectable marker gene is expressed at a consistent level for multiplecell generations, then a cell line results.

One common method for transfection into mammalian cells in particular iscalcium phosphate precipitation. Another method is polyethylene glycol(PEG)-induced fusion of bacterial protoplasts with mammalian cells.Schaffner et al. (1980) Proc. Natl. Acad. Sci. USA 77:2163. Yet anothermethod is electroporation, which can also be used to introduce DNAdirectly into the cytoplasm of a host cell, as described, for example,in Potter et al. (1988) Proc. Natl. Acad. Sci. USA 81:7161.

Transfection of DNA can also be carried out using polyliposome reagentssuch as Lipofectin and Lipofectamine (available from Gibco BRL,Gaithersburg, Md.) which form lipid-nucleic acid complexes (orliposomes), which, when applied to cultured cells, facilitate uptake ofthe nucleic acid into the cells.

Once a cell expressing the desired protein is identified, it can beexpanded and selected. Transfected cells may be selected in a number ofways. For example, cells may be selected for expression of thepolypeptide of interest. For cells in which the vector also contains anantibiotic resistance gene, the cells may be selected for antibioticresistance, which positively selects for cells containing the vector. Inother embodiments, the cells may be allowed to grow under selectiveconditions.

Once a clone producing a protein is identified, the line can be furtherscreened to identify subclones having one or more desired phenotypes,such as but not limited to cells that exhibit high-titer expression,enhanced growth properties, and/or the ability to yield proteins withdesired biochemical characteristics, for example, due to proteinmodification and/or altered post-translational modifications. Thesephenotypes may be due to inherent properties of a given subclone or tomutagenesis. Mutagenesis can be effected through the use of chemicals,UV-wavelength light, radiation, viruses, insertional mutagens, defectiveDNA repair, or a combination of such methods.

Another aspect of the invention features pharmaceuticals composition ofantibodies of the invention. The pharmaceutical compositions may be usedto treat a disease, for example, cancer, an infectious disease, or aninflammatory disease in a patient.

The invention provides pharmaceutical compositions comprising one ormore MAbs for the treatment of disease, such as but not limited tocancer, an infectious disease, or an inflammatory disease, formulatedtogether with a pharmaceutically acceptable carrier.

In prophylactic applications, pharmaceutical compositions areadministered to a patient susceptible to, or otherwise at risk of adisease or condition (e.g., cancer, an infectious disease, or aninflammatory disease) in a prophylactically effective amount. At-riskindividuals include, but are not limited to, individuals with a familyhistory of cancer, an infectious disease, or an inflammatory disease,individuals who have previously been treated for cancer, an infectiousdisease, or an inflammatory disease, and individuals presenting anyother clinical indicia suggesting that they have an increased likelihoodof developing cancer, an infectious disease, or an inflammatory disease.Alternatively stated, an at-risk individual is any individual who isbelieved to be at a higher risk than the general population fordeveloping cancer, an infectious disease, or an inflammatory disease.The term “prophylactically effective amount” is meant to refer to anamount of a formulation which produces an effect observed as theprevention of the onset or recurrence of cancer, an infectious disease,or an inflammatory disease. Prophylactically effective amounts of aformulation are typically determined by the effect they have compared tothe effect observed when a second formulation lacking the active agentis administered to a similarly situated individual.

In therapeutic applications, compositions are administered to a patientsuspected of, or already suffering from such a disease in atherapeutically effective amount sufficient to cure, or at leastpartially arrest, the symptoms of the disease (biochemical and/orhistological), including its complications and intermediate pathologicalphenotypes in development of the disease.

In both prophylactic and therapeutic regimes, agents are usuallyadministered in several dosages until a sufficient response has beenachieved. Typically, the response is monitored and repeated dosages aregiven if the response starts to wane.

Effective doses of a monoclonal antibody for the treatment of disease,e.g., cancer, an infectious disease, or an inflammatory disease, asdescribed herein, vary depending upon many different factors, includingmeans of administration, target site, physiological state of thepatient, whether the patient is human or an animal, other medicationsadministered, and whether treatment is prophylactic or therapeutic.Usually, the patient is a human but nonhuman mammals can also betreated.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from days to severaldays to several months, or until a cure is effected or a diminution ofthe disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of apatient or subject. Persons of ordinary skill can easily determineoptimum dosages, dosing methodologies and repetition rates. Optimumdosages can vary depending on the relative potency of individualantibodies and, in the case of concomitant administration, the relativepotency of known drugs used in the treatment of disease. Optimum dosagescan generally be estimated based on EC50s found to be effective in invitro and in vivo animal models.

In general, dosage is from 0.01 μg to 100 g per kg of body weight andcan be given once or more daily, weekly, monthly or yearly, or even onceevery 2 to 20 years. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Although individual needs can vary, determination of optimal ranges foreffective amounts of formulations is within the skill of the art. Humandoses can be extrapolated from animal studies (REMINGTON'SPHARMACEUTICAL SCIENCES, 20TH ED., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 2000). Generally the dosage required to provide aneffective amount of a formulation, which can be adjusted by one skilledin the art, will vary depending on the age, health, physical condition,weight, type and extent of the disease or disorder of the recipient,frequency of treatment, the nature of concurrent therapy (if any) andthe nature and scope of the desired effect(s).

Pharmaceutical compositions of the invention may be formulated with apharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers include water, PBS, salt solution (such as Ringer'ssolution), alcohols, oils, gelatins, and carbohydrates, such as lactose,amylose, or starch, fatty acid esters, hydroxymethylcellulose, andpolyvinyl pyrrolidine. Such preparations can be sterilized, and ifdesired, mixed with auxiliary agents such as lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, and coloring. Pharmaceutical carriers suitable foruse in the present invention are known in the art (REMINGTON'SPHARMACEUTICAL SCIENCES, 20TH ED., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 2000).

The pharmaceutical formulations, which can conveniently be presented inunit dosage form, can be prepared according to conventional techniqueswell known in the pharmaceutical industry. Such techniques include thestep of bringing into association the active ingredients with thepharmaceutical carrier(s). In general the formulations are prepared byuniformly and intimately bringing into association the activeingredients with liquid carriers or finely divided solid carriers orboth. The formulations can be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and can be stored ina frozen or freeze-dried (lyophilized) condition requiring only theaddition of sterile liquid carrier immediately prior to use.

The pharmaceutical compositions are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Additional guidance regarding formulation, dose and administrationregimen is available in the art (Berkow et al., 1997, THE MERCK MANUALOF MEDICAL INFORMATION, Home, ed., Merck Research Laboratories,Whitehouse Station, N.J.; Goodman et al., 1996, GOODMAN & GILMAN'S THEPHARMACOLOGICAL BASIS OF THERAPEUTICS, 9th ed. McGraw-Hill HealthProfessions Division, New York; Ebadi, 1998, CRC DESK REFERENCE OFCLINICAL PHARMACOLOGY, CRC Press, Boca Raton, Fla.; Katzung, 2001, BASIC& CLINICAL PHARMACOLOGY, 8TH ED. Lange Medical Books/McGraw-Hill MedicalPub. Division, New York; Speight et al., 1997, AVERV'S DRUG TREATMENT: AGUIDE TO THE PROPERTIES, CHOICE, THERAPEUTIC USE AND ECONOMIC VALUE OFDRUGS IN DISEASE MANAGEMENT, 4TH ED. Adis International,Auckland/Philadelphia, Pa.).

When used as a pharmaceutical treatment, the compositions of the presentinvention can be administered either alone or co-administered with othercompounds or compositions that are used in the treatment of disease, forcancer, an infectious disease, or an inflammatory disease. Examples ofsuch compounds, referred to herein as “supplemental compounds,” or“supplemental compositions,” include, but are not limited to,antibiotics, anti-cytokines, anti-asthma drugs, antiphospholipases(e.g., inhibitors of phospholipase), vasodilators (e.g., adenosine,beta-adrenergic agonists or antagonists, β-adrenergic blockers,α-adrenergic blockers, diuretics, smooth muscle vasodilators, nitrates,and angiotensin-converting enzyme inhibitors), biomolecules, cytostaticagents, and chemotherapeutic agents. Pharmaceutical compositions of theinvention may comprise, for example, one or more supplemental compounds.In some embodiments, the antibody is conjugated to the supplementalcompound.

According to yet another aspect of the invention, kits are provided, forexample, for the treatment of cancer, an infectious disease, or aninflammatory disease.

The kits of the invention comprise antibody or an antibody compositionof the invention and instructions for using the kit in a method fortreating cancer, an infectious disease, or an inflammatory disease in apatient or for inhibiting the biological activity of target antigen(e.g., GM-CSF). The kit may comprise at least one supplemental compound.The kit may comprise instructions and/or means for administering theantibody or antibody composition, for example, by injection.

Antibodies of the invention may be used to detect antigen in abiological sample such as but not limited to blood serum. Any methodknown in the art may be used, such as but not limited to flow cytometry.For example, a biological sample may be incubated with antibody of theinvention followed by washing and incubation with a labelled secondaryantibody. For example, the secondary antibody may be directed to thelight chain and conjugated to either FITC or phycoerythrin for purposeof detection.

Antigen neutralizing activity of antibodies of the invention may betested in an antigen neutralization assay by any method known in theart. For example, neutralization activity of the antibodies may beassessed using antigen-dependent cell lines. Examples ofGM-CSF-dependent cell lines include but are not limited to TF-1 andAML-193.

In a first antigen neutralization assay, antigen-dependent cells aresuspended in assay medium, assay medium, antigen, or antigenpre-incubated for one hour with test or isotype control antibodies.Following the incubation period, growth inhibition is evaluated by anymethod known in the art. For example, Cell Titer reagent (Promega, WI)may be added followed by further incubation and measurement of opticaldensity (O.D.) at 490 nm in a spectrophotometer and subtraction ofmedium background from samples. Percentage of antigen neutralization iscalculated as follows: 100−[O.D. with Ig/O.D. without Ig)×100].

In another neutralization assay, antigen is mixed with anti-antigenantibodies of the invention. Antigen-dependent cells are added to themixture, followed by incubation. After this incubation period, growthinhibition is measured. For example, the DNA proliferation marker MTSmay be added followed by measurement of dye incorporation. Decreased dyeincorporation in the presence of antibody relative to that in theabsence of the anti-antigen antibody indicates neutralization ofantigen.

Alternatively, antigen-dependent cells are grown in the presence ofantigen followed by addition of increasing amounts of antibody to theculture media and assessment of neutralizing activity as describedabove.

Therapeutic methods of the invention include methods of inhibiting thebiological activity of a target antigen, for example, GM-CSF and methodsof treating a disease such as but not limited to cancer, an infectiousdisease, or an inflammatory disease by administering the pharmaceuticalcompositions of the antibodies of the invention to a patient or subjectin need thereof. Biological activity of GM-CSF includes but is notlimited to binding to the GM-CSF receptor. The methods may be employed,for example, to effect prophylactic or therapeutic treatment of adisease such as, but not limited to, cancer, an infectious disease, oran inflammatory disease.

The therapeutic methods of the invention are suitable for use in humansand non-human animals. Non-human animals which benefit from theinvention include pets, exotic (e.g., zoo animals) and domesticlivestock. Preferably the non-human animals are mammals.

The antibodies for use in the invention may be administered orally inany acceptable dosage form such as capsules, tablets, aqueoussuspensions, solutions or the like. The antibodies may also beadministered parenterally. That is via the following routes ofadministration: subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intranasal, topically,intrathecal, intrahepatic, intralesional, and intracranial injection orinfusion techniques. Generally, the antibodies will be provided as anintramuscular or intravenous injection.

The antibodies of the invention may be administered alone or with apharmaceutically acceptable carrier, including acceptable adjuvants,vehicles and excipients.

The antibodies of the invention may be administered before, after, orsimultaneously with another therapeutic agent. For example, theantibodies of the invention may be administered alone or co-administeredwith a supplemental compound.

The antibodies of the invention may be administered as a homogenousmixture of unconjugated or conjugated antibody or as a heterogeneousmixture of unconjugated and conjugated antibody.

Effective treatment may be assessed in a variety of ways. In oneembodiment, effective treatment of cancer, an infectious disease, or aninflammatory disease is determined by a slowed progression of thedisease. In still other embodiments, effective therapy is measured byincreased well-being of the patient including such signs as weight gain,regained strength, decreased pain, thriving, and subjective indicationsfrom the patient of better health.

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.

Example 1 Generation of Antigen-Specific Human MAbs to GM-CSF

Materials and Methods

Human B-cells, ex-vivo immunization and cell culturing. In allprocedures followed, cells were grown in 5% CO₂ at 37° C. Leukopackswere obtained from tetanus toxoid (TT)-vaccinated healthy individuals.PBMCs were purified by Ficoll-Plaque (Amersham BioSciences) and CD19positive B-cells and CD4 positive T-cells were isolated from PBMCs byEasySep® human CD4 and CD19 selection kit (StemCell Technologies),respectively, and mixed to make a B-cell/T-cell pool (BT4 cells). BT4cells were cultured in complete RPMI1640 (Invitrogen, CA), whichcontained 10% heat-inactivated human serum AB (Nabi, Fla.), 2 mML-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 55μM 2-mercaptoethanol (Invitrogen, CA).

For ex-vivo immunizations, BT4 cells were co-cultured in the presence ofT- and B-cell epitopes. Briefly, BT4 cells were seeded at a density of10⁶/mL in complete RPMI containing 1 Lf/mL of tetanus toxoid (TT)(Cylex, MD) in the presence of irradiated autologous PBMCs at a 1:1ratio to produce activated T cells (T-pool). To generateantigen-activated B-cells (B-pool), BT4 cells were seeded at a densityof 3×10⁶ cells/mL in complete RPMI containing 10% human serum AB, 5%condition medium from activated T-cells, 20 U/mL IL-2, 0.5 ng/mL IL-6,100 U/mL IL-10 (PrepoTech, NJ), and 250 ng/mL of a cocktail of peptides(bio-World, OH) representing various regions of the targeted antigen,synthesized to contain both T- and B-cell epitopes as previouslydescribed (Zafiropoulos et al. (1997) J. Immunol. Methods 200:181-90).

The selected B-cell epitopes for GM-CSF were: EHVNAIQEARRLLNL (SEQ IDNO:3), STQPWEHVNAIQEAR (SEQ ID NO:4), MASHYKQHCPPTPET (SEQ ID NO:5).

T- and B-pools were separately cultured for 7 days and then co-cultured(10⁶ cell/mL) at a 1:10 ratio, respectively, on a monolayer ofirradiated CHO feeder cells, in complete RPMI containing 10%heat-inactivated human AB serum and 400 U/ml IL-4 (PeproTech, NJ). Afterfive days, co-cultured T- and B-pools were fused to generate hybridomasas described.

For the immortalization of pulmonary alveolar proteinosis (PAP)patients' B-cells, 100 mL of whole blood was processed to purify PBMCs.Lymphocytes were cultured for 7-10 days in complete RPMI containing 10%heat inactivated fetal bovine serum (FBS), (JRH Biosciences, KS), 2ng/mL IL-4 (PeproTech, NJ), 2 mM L-glutamine, 0.1 mM non-essential aminoacids, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol (Invitrogen, CA),50 μg/mL transferrin, 5 ng/mL phorbol myristate acetate (PMA), and 0.5μg/mL cyclosporine A (Sigma, Mo.), in the presence of irradiated CHOfeeder cells. Lymphocytes were then electro-fused as described below.

Cell fusion and Enzyme-Linked Immunosorbent Assay (ELISA) screening ofantigen-reacting hybridomas. Human B-cells used for the generation ofMAbs designed for administration to humans may represent a potentialvehicle of viral transmission. Fusion partner cells and PBMCs fromhealthy donors were pre-screened to confirm absence of viral DNA by PCR,including immunodeficiency-1 and 2, hepatitis B and C, cytomegalo-,herpes-6, and Epstein Barr viruses. Lymphocytes were fused with K6H6/B5cells (ATCC, VA) using the CYTOPULSE CEEF-50 apparatus (Cyto PulseSciences, Inc., MD) at 1:1 lymphocytes to K6H6/B5 ratio.

After fusion, cells were seeded in flat-bottom 96-well microplates at5,000 cells/well in complete RPMI containing 10% heat inactivated FBS, 2mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate,55 μM 2-mercaptoethanol (Invitrogen, CA), 100 μM hypoxanthine, 0.4 μMaminopterin, and 16 μM thymidine (HAT, Sigma, Mo.). Medium was replacedweekly and HAT selection was maintained until completion of antigenreactivity screening (3-5 weeks).

For the identification of antigen-reacting MAbs, ELISA-based screeningswere performed robotically using a BIOMEK FX liquid handling systemintegrated with plate washer and spectrophotometer. Briefly, microtiterplates were coated at room temperature for 6 hours with 50 μL/well ofin-house produced recombinant antigen (1 μg/mL GM-CSF) diluted incoating buffer (50 mM carbonate-bicarbonate, pH 9.4). Plates were thenblocked with binding buffer (PBS containing 1% BSA (Sigma, Mo.) and0.05% Tween 20 (BioRad, CA)) for 2 hours at room temperature. Plateswere washed once with washing buffer (PBS containing 0.05% Tween 20) and50 μL/well of hybridoma supernatant was transferred into the ELISAplates. Binding reaction was carried out at room temperature for 2hours. Subsequently, plates were washed 4 times and 100 μL ofhorseradish peroxidase (HRP)-conjugated goat anti-human IgG+M (JacksonImmunoResearch Laboratories, PA) diluted 1:10,000 in binding buffer wasadded and reactions carried out at room temperature for 1.5 hours.Finally, plates were washed 4 times and 100 μL/well of SureBluesubstrate (KPL, MD) was added for 10 min. Reactions were stopped byadding 50 μL/well of 1 N sulfuric acid and the absorbance was determinedat 450 nm.

Fluorescent-activated cell sorting (FACS) analyses. Ig binding and cellwashing steps were conducted using ice-cold binding buffer (DPBS)without calcium or magnesium, 0.5% BSA), reactions were set up inV-bottom microplates, and samples were analyzed using a FACSAriaapparatus (BD Biosciences, NJ). For the FACS experiment shown in FIG.3A, 10⁶ murine anti-GM-CSF hybridoma cells (Mul19/2) were loaded with100 ng/reaction of GM-CSF and then incubated with anti-GM-CSF human MAbE5. The binding of GM-CSF specific MAbs were detected with 10 μg/mLFITC-labeled goat anti-human Ig (SouthernBiotech, AL). For the FACSexperiment shown in FIG. 3B, the E10 hybridoma cells were washed andseeded at 500,000 cells/well in a volume of 90 μL. Ten μL ofphycoerythrin (PE)-labeled GM-CSF (R&D System, MN) was then added ineach well and cells were incubated on ice for one hour. For unlabeledGM-CSF competition, the hybridoma cells were pre-incubated with 5 μg/mLrecombinant human GM-CSF (PeproTech, NJ) at room temperature for onehour, washed three times, and then incubated with PE-GM-CSF as abovebefore analysis. For the FACS experiment shown in FIG. 4, A431 andA431-K5 cells (gift of Dr. Ira Pastan, National Cancer Institute) werestained with 10 μg/mL of C12 MAb or normal human IgM (JacksonImmunoResearch Laboratories, PA) diluted in binding buffer and reactionswere carried out as above.

GM-CSF neutralization bioassay. The GM-CSF-dependent humanerythroleukemia cell line TF-1 (ATCC, VA) was grown in complete RPMI1640 (see above) containing 10 ng/mL recombinant human GM-CSF(PeproTech, NJ). On the day preceding the experiment, TF-1 cells weregrown in 0.1% FBS in the absence of GM-CSF. Starved cells were washedtwice, resuspended in assay medium, and seeded in 96-well microplates ata concentration of 10,000 cell/well. Wells contained either assaymedium, 100 pg/mL GM-CSF, or GM-CSF pre-incubated for one hour with testor isotype control Ig at concentrations indicated in the figure legends.After 3 days, 40 μL of Cell Titer reagent (Promega, WI) was added toeach well, and plates were further incubated at 37° C. for 1 hour.Optical density (O.D.) was measured at 490 nm in spectrophotometer andmedium background was subtracted to all samples. Percentage of GM-CSFneutralization was calculated as follows: 100−(O.D. with Ig/O.D. withoutIg×100).

Antibody class-switch. Hybridoma cells were washed once with 10 mL PBS,resuspended in complete RPMI, seeded into flat bottom 96-wellmicroplates and incubated at 37° C. in 5% CO₂. After four days, cellswere resuspended by pipetting and 100 μL transferred to 20 ELISPOTplates (Millipore, MA) coated with 2.3 μg/mL goat anti-human IgG (H+L)(Jackson Immunoresearch, PA). The remaining cells in the tissue plateswere fed with an additional 100 μL complete RPMI. After overnightincubation, ELISPOT plates were washed three times with PBS containing0.05% Tween (PBST), then 100 μL of 2 μg/mL goat anti-human IgG (H+L)-HRPwas added and the plates incubated one hour at room temperature withshaking. Plates were washed three times with PBST then 100 μL of AECsubstrate solution (Sigma, St. Louis, Mo.) was added to wells andincubated 90 minutes at room temperature with shaking. Substrate wasaspirated and plates were washed with dH₂O and allowed to air dry.Clones from wells exhibiting positive spots (indicating IgG production)were expanded. The above step was sequentially repeated by reseedingpositive clones at 1000, 100, 10, and 0.25 cells/well while trackingpositive wells until a single-cell colony was identified that secretedIgG.

Fermentation using hollow fibers and stirred bioreactor. Cells wereseeded at 2.5×10⁵/mL in a 2 L bioreactor (B Braun Stat B-DU) containing1 L HyQCDM4NS0 serum free medium (HyClone, UT) maintaining glucose andglutamine at 6 g/L and 4 mM respectively. Controlled set points were: pH7.1, dO₂ 40% saturation with air, temperature 37° C., and agitationrates at 80 rpm. Two mLs of sample was harvested daily, 1 mL for cellcounting using a Cedex apparatus, and 1 mL used to measure Igconcentrations by ELISA. For the hollow fiber run, 108 viable cells wereseeded in a FiberCell system (Bellco, NJ) containing 15 mL of completeRPMI and re-fed using an inline reservoir containing one liter of freshmedium when 50% of the glucose was consumed.

Mismatch repair inhibition to increase genetic diversity of hybridomalines. Hybridoma cells were grown in complete RPMI (negative control) orcomplete RPMI containing 250 μM or 500 μM of MMR-inhibiting anthracenecompound. Cells were passed at a 1:5 dilution every three to four daysin fresh media with or without morphocene and after three weeks cellswere harvested and resuspended at 2×10⁶ cells/mL in FACS buffer (PBSwith 1% BSA). Cells were stained with 10 μg/mL FITC-conjugated goatanti-human Ig (Jackson Immunoresearch) for 30 minutes on ice. Cells werewashed with 10 mL ice cold FACS buffer and resuspended in 3 mL FACSbuffer. 10 μL Viaprobe (Becton Dickinson, Franklin Lakes, N.J.) wasadded for 5 minutes on ice and viable cells were sorted for high Igsurface staining on a FACSAria cell sorter (Becton Dickinson). The gatewas set to sort cells representing the 5% subpopulation with the highestIg surface staining. For selection of clones with enhanced titers, FACSsorted cells were seeded in U-bottom 96 well plates and incubated forone week at 37° C. in 5% CO₂. Fifty μL of supernatants were harvestedfrom wells and analyzed for IgM production via ELISA using goatanti-human IgM+G coated plates. As an internal control, 3 wells of eachELISA plate were seeded with 50 μL of 10 ng/mL human IgM (JacksonImmunoresearch). O.D. values obtained at 450 nm were normalized to themean values of internal control wells. Wells exhibiting high IgM signalswere expanded for further analysis. For microsatellite instability (MSI)analysis, DNA was extracted from parental or morphocene-treated cellsusing the Qiagen DNeasy Tissue kit (Qiagen). The BAT poly A repeatmarker (7) was amplified using the D4 fluorescent-labeled BAT-26-F(5′-tcaccatccattgcacagtt-3′) (SEQ ID NO:20) and BAT-26-R(5′-ctgcgagaaggtactcaccc-3′) (SEQ ID NO:21) primers, pfuUltra™high-fidelity polymerase (Stratagene, CA), and reactions incubated asfollows: 5 min. at 95° C.; 9 cycles of 1 min. at 94° C., 1 min. at 60°C. and 2 min. at 72° C., with the annealing temperature decreasing by 1°C. each cycle; 30 cycles of 1 min. at 94° C., 1 min. at 52° C., and 2min. at 72° C.; final extension of 10 min. at 72° C. Single-copies ofthe marker allele were obtained by using a dilution of DNA that yieldedan amplicon in only 50% of the PCR reactions. PCR products were diluted1:10 with CEQ sample load solution and then loaded into the Beckman CEQ8000 Genetic Analysis System for fragment analysis.

Generation of antigen-specific human MAbs. Ex-vivo immunizations werecarried out using cryo-preserved B-cells obtained from volunteersubjects (healthy donors) as described above. Alternatively, B-cellswere obtained from human subjects whose sera contained high titers ofMAbs specific to an antigen of interest. The rationale of the latterapproach stems from the possibility that some antigen-specific MAbscould result from an abnormal immune response (as in the case ofautoimmune patients), or derive from an in vivo immune response totumor, microbial, or vaccine antigens.

Several antigen-reacting human MAbs were identified after fusion ofex-vivo immunized cells and from hybridoma libraries generated from PAPpatients B-cells. Four hybridoma lines, E5 (IgM), G7 (IgM), E10 (IgG),and G9 (IgG) were selected for further studies and the human MAbs theyproduce were tested for specificity by ELISA. FIG. 1 shows that E5, G7,and E10 human MAbs only reacted with human GM-CSF and none of the otherten unrelated antigens tested, including murine GM-CSF, which shares a53% identity with the human homolog. Similar results were obtained forthe G9 hybridoma. FACS analyses were carried out to confirm specificityof these human MAbs. Human GM-CSF was allowed to bind to the surface ofmouse hybridoma cells, which express membrane bound-MAbs specific tohuman GM-CSF at a different epitope. E5 MAb bound the surface of thesecells under these conditions, as indicated by the fluorescence intensityshift (FIG. 2A, bottom panel). This result demonstrates the ability ofE5 MAb to bind native human GM-CSF. In absence of cell-bound GM-CSF, E5MAb did not cross-react with any of the membrane bound proteinsexpressed by these hybridoma cells (FIG. 2A, middle panel). Similarly,E10 MAb showed high specificity via FACS analysis. In addition, sinceE10 MAb was found associated to the hybridoma cell membrane, its abilityto bind soluble, phycoerythrin (PE)-labeled GM-CSF was shown by FACS(FIG. 2B, middle panel). Binding specificity was demonstrated bypre-incubation of the E10 hybridoma cells with an excess of unlabeledGM-CSF (FIG. 2B, lower panel).

De novo class-switch of human MAbs. Using the two strategies describedabove, IgG and IgM human MAbs to a variety of human and nonhumanantigens have been generated. Although most therapeutic antibodies inthe market are of the IgG isotype, cancer trials testing potentiallytherapeutic IgM MAbs have shown regression of tumors in vivo (16, 17).These clinical responses can be attributable to the ability of IgM tostrongly fix and activate the complement pathway and effectively killtumor cells. IgG binds to the Fc receptors on macrophages and NK cellsand thus can mediate ADCC activity against tumor cells. Both IgG and IgMwith identical specificity (same antigen and epitope) can be tested forbest pharmacological activity in vivo. In the case where an IgG isotypeis preferred, a quick robust procedure (see Materials and Methods) forde novo class-switching of IgM has been followed. Using the E5 line asan example, a subset of cells that had class-switched to an IgG isotypeunder the growth conditions used was identified. The E5 IgG showedidentical nucleotide sequence in its variable region and similarreactivity to GM-CSF (FIG. 3) as the parental E5 IgM.

Biological activities of human MAbs. Pharmacological properties soughtfor therapeutic MAbs that target soluble mediators of disease includethe ability to neutralize growth factors. As mentioned above, one suchexample is GM-CSF as a mediator of RA (9-11). The ability of the humanMAbs to block GM-CSF function using a cell-based assay whereby thegrowth of human erythroblastoid cells (TF1) is dependent on the presenceof this cytokine in their culture medium was assessed. As shown in FIG.4, both E10 and G9 significantly inhibited GM-CSF-dependent cell growth,whereas the human IgG isotype control showed no effect. The differencein potency seen between E10 and G9 correlates well with their apparentaffinities of 870 and 14 picomolar, respectively. The E5 MAb only showedminimal neutralizing activity consistent with its lower affinity (5 nM).

Assessment of titers and stability of hybridomas secreting human MAbs.An important property of a MAb manufacturing line is stability of Igsecretion during the entire batch manufacturing cycle. In one scenariowhere the cycle duration is about two months, a line doubling every 24hours would go through about 60 generations from thawing to harvest. AnE5 line was used as a model for testing MAb titers and productionstability of hybridoma generated using our method. A clone derived fromthis line, 3D2, showed a doubling time of 24 hours and was re-cloned bylimiting dilutions after more than two months of continuous culturing.The frequency of producing clones was determined via ELISA, measuring Igconcentrations in their conditioned media normalized for cell densities.FIG. 5A shows that all E5-3D2 subclones tested secrete high levels ofIg, demonstrating homogeneous retention of Ig production in this cellpopulation after 60 generations. Ig production was then assessed using asmall scale (15 mL) hollow fiber system. Cells were inoculated in ahollow fiber cartridge and continuously fed using an inline reservoircontaining one liter of fresh medium. Starting on day 5, all conditionedmedium from the cartridge (15 mL) was harvested daily and replaced withfresh medium. Fermentation was carried out for additional 4 days, whiledaily Ig titers were determined by ELISA using an Ig standard of knownconcentration. A cumulative titer of 1.2 g/L during the 4-day run wasrecorded. Between day 8 and 9, glucose consumption was at its peak (2gram/L a day), indicating that cells tolerated well the extremely highcell densities. Production performance was also evaluated in a 1-literscale fed-batch run using a stirred bioreactor system. Cells from afrozen ampule were first thawed and inoculated in a shake flask andlater seeded in stirred bioreactor (Bauer) containing 1 liter ofserum-free medium. Fermentation was carried out until cell viabilitydropped below 60% (day 6). Ig production and cell densities wererecorded between day 1 and 5 and are shown in FIG. 5B. During the logphase (day 1-4), a specific productivity of 24 pg/cell/day with adoubling time averaging 23.4 hours was measured, suggesting goodscalability of these cells from flask to bioreactor while maintaininghigher titers.

Genetic optimization of hybridoma secreting human MAbs via mismatchrepair regulation. The usefulness of improving the quality ofMAb-producing cell lines using a process, termed morphogenics, whichentails the transient regulation of MMR has been previously demonstrated(Nicolaides et al. (1995) Genomics 30:195-206; and, Nicolaides et al.(1998) Mol. Cell. Biol. 18:1635-41). After increasing the geneticdiversity of the cell pool using this method, high throughput screeningswere carried out to identify subclones exhibiting higher titer, affinity(Grasso et al. (2004) Bioprocess Int. 2:58-64; and, Nicolaides et al.(2005) Ann. N.Y. Acad. Sci. 1059: 1-11), or enhanced growth rates(Grasso, L. personal observation). E5 cells were subjected tomorphogenics to demonstrate the ability to increase phenotypic diversityin the MAb-secreting lines generated using our hybridoma strategy. MMRinhibition was monitored by detecting microsatellite instability (MSI)in the BAT poly-A repeat marker. Of the 24 BAT alleles analyzed in cellsexposed to the morphogenics process, 3 alleles showed alterations thatincluded single nucleotide deletions, shown in FIG. 6A, and insertions.No MSI was detected in any of the 24 BAT alleles in parental cells.Subsequently, parental or morphogenics-treated cells were seeded bylimiting dilutions in microplates. Cell clones were allowed to secretMAbs for one week and their conditioned medium analyzed for Igconcentrations by ELISA. The frequency of clones with O.D. greater than1 (high Ig secretion) was determined from the total number of clonesscreened (3,763 for parental and 2,437 for morphocene pool) and found tohave increased by 260% (p=0.0014) in the morphogenics-treated population(FIG. 6B).

Summary

This study represents a viable strategy for developing human MAbs forimmunotherapies using an optimized ex-vivo immunization and human B-cellimmortalization process combined with inhibition of mismatch repair.With this approach, highly specific and biologically active MAbssecreted by stable hybridoma lines can be generated.

According to the methods of the invention, stable MAb production forover 60 doublings and production of over 1 gram of MAb per liter duringa 4-day hollow fiber fermentation run has been achieved, suggesting thathybridoma cells generated by the present methods are suited forperfusion systems and potentially large scale manufacturing. Moreover,hybridomas generated by this process have performed well in fed-batchbioreactor runs, suggesting a potential use of these lines forcommercial applications. In summary, the platform process presented hereoffers an alternative approach for a rapid and cost-effectivedevelopment of good quality, fully human antibodies forimmunotherapeutic use.

Example 2 Generation of Fully Human Anti-GM-CSF Antibody; Isolation ofPAP Cells

Patients with adult human pulmonary alveolar proteinosis (PAP)accumulate phospholipids and surfactant proteins in the alveoli. It hasbeen hypothesized that PAP is due to the inability of the alveolarmacrophages and type II epithelial cells to clear excess surfactant. Asdescribed above, the role for GM-CSF in the control of lung surfactanthomeostasis has been established in the murine model and by extension iscausative of the human pathology. Furthermore, patients with PAP havebeen shown to have circulating, neutralizing antibodies to GM-CSF,thereby implicating this cytokine as causative of the disease. Whetherthis autoimmune response is specific for GM-CSF is unclear. However, ithas been shown that a subset of PAP patients improve with GM-CSFtherapy, supporting the hypothesis that the absence of GM-CSF either bygene disruption or antibody-mediated neutralization results in thedevelopment of PAP.

Isolation of GM-CSF-specific antibodies. Peripheral blood mononuclearcells (PBMCs) were isolated from PAP patients. Briefly, the PAPpatient's B cells were recovered from the whole blood. The whole bloodwas diluted with equal volume PBS−/− and the contents was mixed gentlyby inverting the container. 25 ml diluted blood was overlayed onto a 50ml tube that contained 25 ml Ficoll-Paque (Amersham Biosciences AB,Uppsala Sweden). The tubes were centrifuged at 2,000 rpm for 30 mins atroom temperature. PBMCs were collected from the interface layer using a10-ml pipette, transferred to new 50-ml tubes, and washed twice withPBS−/−. The PBMC pellet was re-suspended in 10 ml ACK Lysing Buffer (150mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA, pH 7.2), incubated for 5 min. atroom temperature to lyse the red cells, and washed twice with PBS−/−.

Fusion of PAP B-Cells with Myeloma Cell Line to Generate Hybridoma.Enriched B-cells from PAP patients were fused with a variety of myelomacells (human-mouse heterohybridoma, ATCC, VA, USA); CBF-7 cells(human-mouse heterohybridoma); HEK293; human myeloma cells by thefollowing method. The B cells and fusion partner cells should have goodviability (≧90% viable and in log phase). Both cell types in theiroriginal medium were counted and mixed in 15 ml tubes at 1:1 ratio,followed by centrifugation at 1,000 RPM for 6 min at 4° C. Cells werewashed 3 times using 10-15 ml cold CPFM (CYTOPULSE fusion medium, CytoPulse Sciences, MD USA). The final pellet was resuspended in 10×10⁶cells/ml CPFM. Electro-fusion was performed using CYTOPULSE CEEF-50(Cyto Pulse Sciences, MD USA). Fusion parameters were optimizedfollowing manufacturer and empirical guidelines, and fusion efficiencyaveraged one hybrid in 5,000 cells pulsed. After fusion, cells wereseeded in flat-bottom 96-well microplates at ˜5,000 cell/well incomplete RPMI containing 10% heat inactivated FBS, 100 μM hypoxanthine,0.4 μM aminopterin, and 16 μM thymidine (HAT, Sigma, Mo. USA). Mediumwas replaced weekly and HAT selection was maintained until completion ofantigen reactivity.

ELISA Screening Assay. For the identification of antigen-reacting MAbs,ELISA-based screenings were performed robotically using a BIOMEK FXliquid handling system integrated with plate washer andspectrophotometer. Briefly, microtiter plates were coated at roomtemperature for 6 hours with 50 μL/well of in-house produced recombinantantigens (1 μg/mL GM-CSF) diluted in coating buffer (50 mMcarbonate-bicarbonate, pH 9.4). Plates were then blocked with bindingbuffer (PBS containing 3% BSA (Sigma, Mo.) and 0.05% Tween 20 (BioRad,CA)) for 2 hours at room temperature. Plates were washed once withwashing buffer (PBS containing 0.05% Tween 20) and 50 μL/well ofhybridoma supernatant was transferred into the ELISA plates. Bindingreaction was carried out at room temperature for 2 hours. Subsequently,plates were washed 4 times and 100 μL of HRP-conjugated goat anti-humanIgG+M (Jackson ImmunoResearch Laboratories, PA) diluted 1:10,000 inbinding buffer was added and reactions carried out at room temperaturefor 1.5 hours. Finally, plates were washed 4 times and 100 μL/well ofSUREBLUE substrate (KPL, MD) was added for 10 min. Reactions werestopped by adding 50 μL/well of 1 N sulfuric acid and the absorbance wasdetermined at 450 nm.

FACS Analysis and Sorting. In every study, Ig binding and cell washingsteps were conducted using ice-cold binding buffer (DPBS without calciumor magnesium, 0.5% BSA). The PBMC or hybridoma cells were washed andseeded at 500,000 cell/well. The FITC and phycoerythrin(PE)-labeledanti-human CD3, CD 19, CD20 (SouthernBiotech, AL), and PE-labeled GM-CSF(R&D System, MN) were added (10-100 diluted), and incubated on ice forone hour. The cells were then washed three times with binding buffer,and analyzed or sorted using a FACSARIA apparatus (BD Biosciences, NJ).Results of isotype analysis of Mab E10 are shown in FIG. 9. To determinethe isotype of E 10, a standard analysis was performed using anti-humanIgG, IgG1, IgG2, IgG3, IgM, Lκ, and Lλ Fc specific antibodies to derivethe isotype.

GM-CSF Neutralization Bioassay. The human GM-CSF-dependent humanerythroleukemia cell line TF-1 (ATCC, VA) was grown in complete RPMI1640 (see above) containing 10 ng/mL recombinant human GM-CSF(PeproTech, NJ). On the day preceding the experiment, TF-1 cells weregrown in 0.5% FBS in the absence of GM-CSF. The starved TF-1 cells wereharvested and washed twice with assay medium (plain RPMI with 0.5% BSA).Cells were resuspended in assay medium and seeded in 96-well microplatesat a concentration of 10,000 cells/well. Wells contained either assaymedium, 100 pg/mL GM-CSF, or GM-CSF pre-incubated for one hour with testor isotype control Igs at concentrations indicated in the figure. After3 days, 40 μL of Cell Titer reagent (Promega, WI) was added to eachwell, and plates were further incubated at 37° C. for 1 hour. Opticaldensity (O.D.) was measured at 490 nm in a spectrophotometer and mediumbackground was subtracted from all samples. Percentage of GM-CSFneutralization was calculated as follows: 100-[(O.D. with Ig/O.D.without Ig)×100)]. FIG. 4 demonstrates GM-CSF-dependent growthinhibition of TF-1 cell lines with fully human MAbs.

Inhibition of Mismatch Repair to Increase Genetic Diversity of HybridomaCell Lines. Inhibition of mismatch repair can lead to geneticallydiverse sibling cells with enhanced production, cell growth, or antibodyactivity. To improve MAb activity and cell growth, hybridoma cells weregrown in complete RPMI (negative control) or complete RPMI containing250 μM or 500 uM of the mismatch repair inhibitor morphocene(9,10-dimethyl anthracene, MP Biomedicals, CA). Cells were passed at a1:5 dilution every three to four days in fresh media with or withoutmorphocene and after three weeks cells were harvested and resuspended at2×10⁶ cells/mL in FACS buffer (PBS with 1% BSA). Cells were stained with10 μg/mL FITC-conjugated goat anti-human Ig (Jackson Immunoresearch) for30 minutes on ice. Cells were washed with 10 mL ice cold FACS buffer andresuspended in 3 mL FACS buffer. 10 μL VIAPROBE (Becton Dickinson,Franklin Lakes, N.J.) was added for 5 minutes on ice and viable cellswere sorted for high Ig surface staining on a FACSARIA cell sorter(Becton Dickinson). The gate was set to sort cells representing the 5%sub-population with the highest Ig surface staining. This population wasexpanded for one week in the presence or absence of chemical inhibitorof mismatch repair (MMR) and the procedure repeated two additionaltimes. For selection of clones with enhanced titers, FACS sorted cellswere seeded in U-bottom 96 well plates at 0.8 cells/well in 200 μLcomplete RPMI. Plates were incubated for one week at 37° C. in 5% CO₂.50 μL of supernatants were harvested from wells and analyzed for IgMproduction via ELISA using goat anti-human IgM+G coated plates. As aninternal control, 3 wells of each ELISA plate were seeded with 50 μL of10 ng/mL human IgM (Jackson Immunoresearch). O.D. values obtained at 450nm were normalized to the mean values of internal control wells. Wellsexhibiting high IgM signals were expanded for further analysis. For MSIanalysis, DNA was extracted from parental or MMR inhibitor-treated cellsusing the Qiagen DNeasy Tissue kit (Qiagen). The BAT poly A repeatmarker was amplified using the D4 fluorescent-labeled mBAT-26-F(5′-tcaccatccattgcacagtt-3′) (SEQ ID NO:20) and mBAT-26-R(5′-ctgcgagaaggtactcaccc-3′) (SEQ ID NO:21) primers, pfuUltra™high-fidelity polymerase (Stratagene, CA), and reactions incubated asfollows: 5 min. at 95° C.; 9 cycles of 1 min. at 94° C., 1 min. at 60°C. and 2 min. at 72° C., with the annealing temperature decreasing by 1°C. each cycle; 30 cycles of 1 min. at 94° C., 1 min. at 52° C., and 2min. at 72° C.; final extension of 10 min. at 72° C. Single-copy of themarker's alleles were obtained by using a dilution of DNA that yieldedan amplicon in only 50% of the PCR reactions. PCR products were diluted1:10 with CEQ sample load solution and then loaded into the Beckman CEQ8000 Genetic Analysis System for fragment analysis. (Blake et al.Stepwise deletions of polyA sequences in mismatch repair-deficientcolorectal cancers. (2001) Am. J. Pathol. 158:1867-70.)

Example 3 Generation of Monoclonal Antibody E10 with High Specificity toGM-CSF

Several antibodies were derived from B-cells of patients with PAP. Theseantibodies were of the IgM, IgG isotype. Each of these antibodies werefound to specifically bind to GM-CSF using the assays for specificitydescribed herein. One anti-GM-CSF human IgG1 monoclonal antibody E10 wasgenerated from Pulmonary alveolar proteinosis (PAP) patient's B cellsfused with either myeloma cells then subsequently screened foranti-human GM-CSF monoclonal antibodies by ELISA as described above.

Blood from a PAP patient (91 ml whole blood) was used to isolate a totalof 49.5 million PBMCs. The viability of these cells was 99.0%. Theresults from a FACS analysis are shown in Table 2. The B cells wereexpanded by culturing approximately 25 million PBMCs in cRPM11640 (10ml) with IL-4 2 ng/ml (PeproTech), transferrin 50 ug/ml (Sigma), PMA 5ng/ml (Sigma), and cyclosporine A 0.5 ug/ml (Sigma) with feeder cells.After 11 days of culture, 12 million cells remained and weresubsequently fused with a myeloma cell by electro-fusion (CytoPulseCEEF-50). FACS analysis results are set forth in Table 2.

TABLE 2 Generation of E10 monoclonal antibody and analysis by FACS FITC-CD3(%) FITC-CD20(%) PE-GMCSF(%) CD20/GMCSF PBMC 62.6 14.4 5.9 6.3 11 day77.2 17.0 0.7 0.1 culture

The fused cells were cultured in RPMI 1640 (Invitrogen, CA, USA) with10% FBS, heat inactivated (JRH Biosciences, KS USA); L-glutamine, 200 mM(Invitrogen, CA, USA); non-essential amino acids, 10 mM (Invitrogen, CA,USA); sodium pyruvate solution, 100 mM (Invitrogen, CA, USA); Pen-Strep(Invitrogen, CA, USA); 2-Mercaptoethanol, 55 mM (Invitrogen, CA, USA);and 1×HAT (Sigma, Mo. USA).

After 12 days culture, about 48% of the hybridoma culture showed growth.Next, the hybridomas were screened using a GM-CSF specific ELISA withrecombinant GM-CSF (PeproTech, NJ, USA). Several clones were isolated,including those that were positive for GM-CSF. These clones were testedagain by ELISA to confirm that they were specific for the recombinantGM-CSF and not for tetanus toxin (TT). Clone 4E10 showed specificallyreaction with GM-CSF, but not TT. Clone 4E10 was subsequently subcloned.After 3 weeks the cultured subclones were screened by ELISA to confirmspecificity to GM-CSF was maintained and then further characterized asdescribed below.

Characterization of E10. To determine the specificity of the anti-GM-CSFantibodies E10, an antigen-specific ELISA was performed with a range ofantigens (FIG. 8). FIG. 8 compares binding to the following antigens:hIL-1a, h IL-2, h IL-3, h IL-4, h IL-5, IL-6, hIL-13, hGM-CSF, mGM-CSF,BSA, and TT. In subsequent studies, the binding of anti-GM-CSFantibodies was compared to human GM-CSF, human Mesothilin, SEB, BGG,CAB, HEL, TT, BSA, Goat IgG, human mucin, and Mouse IgG (data notshown). In both studies 4E10 only reacted with human GM-CSF.

To determine the isotype of E10, a standard analysis was performed usinganti-human IgG, IgG1, IgG2, IgG3, IgM, Lκ, and Lλ to derive the isotype.This analysis demonstrated that 4E10 is an IgG1 and Kappa antibody (seeFIG. 9).

The ability of E10 to neutralize the biological activity of GM-CSF invitro was tested by using a cell line, TF-1, that is dependent upon thiscytokine for survival and growth (see FIG. 8). The humanGM-CSF-dependent human erythroleukemia cell line TF-1 (ATCC, VA) wasgrown in complete RPMI 1640 containing 10 ng/mL recombinant human GM-CSF(PeproTech, NJ). On the day preceding the experiment, TF-1 cells weregrown in 0.1% FBS in the absence of GM-CSF. The starved TF-1 cells wereharvested and washed twice with assay medium (plain RPMI with 0.5% BSA).Cells were suspended in assay medium and seeded in 96-well microplatesat a concentration of 10,000 cell/well. Wells contained either assaymedium, 100 pg/mL GM-CSF, or GM-CSF pre-incubated for one hour with testor isotype control Igs at concentrations ranging from 20 μg/mL to 0.315μg/mL. After 3 days, 40 uL of CELL TITER reagent (Promega, WI) was addedto each well, and plates were further incubated at 37° C. for 1 hour.Optical density (O.D.) was measured at 490 nm in spectrophotometer andmedium background was subtracted from all samples. Percentage of GM-CSFneutralization was calculated as follows: 100-[(O.D. with Ig/O.D.without Ig)×100]. The antibody 4E10 was capable of neutralizing theactivity of GM-CSF in vitro at a concentration of 100 pg/ml.

A Western blot analysis was performed with E10 to determine if theantibody would cross-react with human recombinant GM-CSF. As shown inFIG. 10, E10 cross-reacts with human GM-CSF under reducing conditions.

To determine the binding affinity of E10, a BIACORE analysis wasperformed as follows. The binding constant for E10 is about 870 pM (FIG.11).

Example 4 Generation of Monoclonal Antibody G9 with High Specificity toGM-CSF

The anti-GM-CSF human IgG1 monoclonal antibody G9 was generated fromPulmonary alveolar proteinosis (PAP) patient's B cells fused with eitherK6 or CBF-7 then subsequently screened for anti-human GM-CSF monoclonalantibodies by ELISA as described above.

Blood from a PAP patient (91 ml whole blood) was used to isolate a totalof 49.5 million PBMCs. The viability of these cells was 99.0% (data notshown). The B cells were expanded by culturing approximately 25 millionPBMCs in cRPMI1640 (10 ml) with IL-4 2 ng/ml (PeproTech); transferrin 50ug/ml (Sigma); PMA 5 ng/ml (Sigma); and cyclosporine A 0.5 ug/ml (Sigma)with feeder cells. After 11 days of culture, 12 million cells remainedand were subsequently fused to myeloma cells by electro-fusion(CytoPulse CEEF-50).

The fused cells were cultured in RPMI 1640 (Invitrogen, CA, USA) with10% FBS, heat inactivated (JRH Biosciences, KS USA); L-glutamine, 200 mM(Invitrogen, CA, USA); non-essential amino acids, 10 mM (Invitrogen, CA,USA); sodium pyruvate solution, 100 mM (Invitrogen, CA, USA); Pen-Strep(Invitrogen, CA, USA); 2-Mercaptoethanol, 55 mM (Invitrogen, CA, USA);and 1×HAT (Sigma, Mo. USA).

After 12 days culture, about 48% of the hybridoma culture showed growth.Next, the hybridomas were screened using a GM-CSF-specific ELISA withrecombinant GM-CSF (PeproTech, NJ, USA). Several clones were isolated,including those that were positive for GM-CSF. These clones were testedagain by ELISA to confirm that they were specific for the recombinantGM-CSF and not for tetanus toxin (TT). The G9 population showed highlyspecific reactivity with GM-CSF, but not TT. Next, the G9 population wassubcloned to obtain a pure culture. After 3 weeks the cultured subcloneswere screened by ELISA to confirm specificity to GM-CSF was maintainedand then further characterized as described below.

Characterization of G9. To determine the specificity of the anti-GM-CSFantibodies G9, an antigen specific ELISA was performed with a range ofantigens (FIG. 8). FIG. 8 compares binding to the following antigens:hIL-1a, h IL-2, h IL-3, h IL-4, h IL-5, IL-6, hIL-13, hGM-CSF, mGM-CSF,BSA, and TT. In subsequent studies the binding of anti-GM-CSF antibodieswas compared to human GM-CSF, human mesothelin, SEB, BGG, CAB, HEL, TT,BSA, Goat IgG, human mucin, and Mouse IgG (data not shown). In bothstudies G9 only reacted with human GM-CSF.

To determine the isotype of G9, a standard analysis was performed usinganti-human IgG, IgG1, IgG2, IgG3, IgM, Lκ, and Lλ to derive the isotype.This analysis demonstrated that G9 is an IgG1 and Kappa antibody (FIG.12).

G9 Neutralization Bioassay. The ability of G9 to neutralize thebiological activity of GM-CSF in vitro was tested by using a cell line,TF-1, that is dependent upon this cytokine for survival and growth (FIG.4). The human GM-CSF-dependent human erythroleukemia cell line TF-1(ATCC, VA) was grown in complete RPMI 1640 containing 10 ng/mLrecombinant human GM-CSF (PeproTech, NJ). On the day preceding theexperiment, TF-1 cells were grown in 0.1% FBS in the absence of GM-CSF.The starved TF-1 cells were harvested and washed twice with assay medium(plain RPMI with 0.5% BSA). Cells were suspended in assay medium andseeded in 96-well microplates at a concentration of 10,000 cell/well.Wells contained either assay medium, 100 pg/mL GM-CSF, or GM-CSFpre-incubated for one hour with test or isotype control Igs atconcentrations ranging from 20 μg/mL to 0.315 μg/mL. After 3 days, 40 uLof CELL TITER reagent (Promega, WI) was added to each well, and plateswere further incubated at 37° C. for 1 hour. Optical density (O.D.) wasmeasured at 490 nm in spectrophotometer and medium background wassubtracted from all samples. Percentage of GM-CSF neutralization wascalculated as follows: 100-[O.D. with Ig/O.D. without Ig)×100]. Theantibody G9 was capable of neutralizing the activity of GM-CSF in vitroat a concentration of 100 pg/ml.

A Western blot analysis was performed with G9 to determine if theantibody would cross-react with human recombinant GM-CSF. As shown inFIG. 10, G9 cross-reacts with human GM-CSF under reducing conditions.

To determine the binding affinity of G9, BIACORE analysis was performedas described above. The binding constant for G9 is about 11-17 μM (FIG.13).

Example 5 Nucleotide Sequences Encoding Fully Human Anti-GM-CSF AntibodyG9 and E10

Antibody G9. Nucleotide and amino acid sequences for fully humananti-GM-CSF antibody G9 was obtained by standard methods. Briefly, totalRNA was isolated from hybridoma G9 using Trizol reagent (Invitrogen)according to the manufacturer's instructions. The message wassynthesized to cDNA using Superscript II reverse transcriptase(Invitrogen) according to the manufacturer's instructions. To amplifythe light and heavy chain variable regions, PCR reactions were carriedout with Herculase DNA polymerase (Stratagene) using primers SEQ IDNO:22 and SEQ ID NO:23 for the light chain and SEQ ID NO:24 and SEQ IDNO:25 for the heavy chain. PCR products were cloned into pCR4-TOPOvector (Invitrogen), transformed into E. coli Machl cells andtransformants selected on LB Kanamycin plates. Colonies were screenedfor inserts with the same primer pairs as above and four positivecolonies each were used to generate template DNA for DNA sequencedetermination, using TempliPhi reagent (GE Healthcare). DNA inserts weresequenced with primers SEQ ID NO:26 and SEQ ID NO:27 using BeckmanCoulter DTCS sequencing reagent followed by data acquisition andanalysis on a Beckman Coulter CEQ2000. In order to add a leader peptidesequence to the light chain, a positive clone was re-amplified withprimers SEQ ID NO:28 and SEQ ID NO:23 using Herculase DNA polymerase. Togenerate a full length heavy chain (SEQ ID NO:11), including a leaderpeptide sequence, PCR was carried out with primers SEQ ID NO:29 and SEQID NO:30 using the original cDNA as template. The resulting PCR productwas TA cloned, transformed into Machl cells and positive clones wereidentified as described above. Full length G9 heavy chain cDNA wassequenced with primers SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:27 and SEQ ID NO:30 usingtemplate DNA generated with TempliPhi reagent. The resulting DNAsequences for full length heavy chain for G9 (SEQ ID NO:11) and fulllength light chain for G9 (SEQ ID NO:15) are shown below. The predictedtranslation products derived from SEQ ID NO:11 and SEQ ID NO:15 areshown in SEQ ID NO:9 and SEQ ID NO:13, respectively. The predictedtranslation products derived from SEQ ID NO:10 and SEQ ID NO:14 areshown in SEQ ID NO:8 and SEQ ID NO:12, respectively. The underlinedsequences of SEQ ID NOs: 11 and 15 represent the leader sequence addedby PCR. The polynucleotide sequences of SEQ ID NO:10 and 14 encode theheavy and light chains of the G9 antibody, respectively, without theadded leader sequences. The lower case sequences of SEQ ID NOs:9 and 13represent the human leader peptides added by the PCR reaction. Theunderlined sequences of SEQ ID NOs: 8, 9, 12, and 13 represent the CDRregions. The remaining sequences of SEQ ID NOs:8, 9, 12 and 13 are theframeworks of the variable regions and the constant regions. Theconstant region for heavy chain starts with amino acid sequence WGQG(amino acid 111 of SEQ ID NO:8 or 130 of SEQ ID NO:9), and the constantregion for the light chain starts with amino acid sequence FGQG (aminoacid 98 of SEQ ID NO:12 or 117 of SEQ ID NO:13).

Antibody E10. Nucleotide and amino acid sequences for fully humananti-GM-CSF antibody E10 was obtained by standard methods. Briefly,total RNA was isolated from hybridoma E10 using Trizol reagent(Invitrogen) according to the manufacturer's instructions. The messagewas synthesized to cDNA using Superscript II reverse transcriptase(Invitrogen) according to the manufacturer's instructions. To amplifythe light and heavy chain variable regions, PCR reactions were carriedout with Herculase DNA polymerase (Stratagene) using primers SEQ IDNO:22 and SEQ ID NO:23 for the light chain and SEQ ID NO:24 and SEQ IDNO:25 for the heavy chain. PCR products were cloned into pCR4-TOPOvector (Invitrogen), transformed into E. coli Machl cells andtransformants selected on LB Kanamycin plates. Colonies were screenedfor inserts with the same primer pairs as above and four positivecolonies each were used to generate template DNA for DNA sequencedetermination, using TempliPhi reagent (GE Healthcare). DNA inserts weresequenced with primers SEQ ID NO:26 and SEQ ID NO:27 using BeckmanCoulter DTCS sequencing reagent followed by data acquisition andanalysis on a Beckman Coulter CEQ2000. The resulting DNA sequencesencoding the heavy chain variable region for E10 (SEQ ID NO:17) and fulllength light chain for E10 (SEQ ID NO:19) are shown below. The predictedtranslation products derived from SEQ ID NO:17 and SEQ ID NO:19 areshown in SEQ ID NO:16 and SEQ ID NO:18, respectively. The underlinedsequences of SEQ ID NOs: 16 and 18 represent the CDR regions. Theremaining sequences of SEQ ID NOs: 16 and 18 are the framework of thevariable region and the constant regions. The constant region for heavychain starts with amino acid sequence WGQG (amino acid 115 of SEQ IDNO:16), and the constant region for the light chain starts with aminoacid sequence FGQG (amino acid 98 of SEQ ID NO:18).

Example 6 Epitope Mapping of Anti-GM-CSF Antibody

To map the epitope of GM-CSF to which G9 binds, a series of overlappingpeptides were generated that span the length of human GM-CSF (FIG. 14).Briefly, twenty-five overlapping 12-mer peptides encompassing the humanGM-CSF sequence (GenBank Accession #AAA52578, residues 14 to 144) weredesigned to contain 7 amino acid overlaps. Peptides were generated asindividual 3.7 mm×3.7 mm spots by solid phase synthesis via attachmentof the C-terminus of each peptide to the surface of a derivatizedcellulose membrane (SPOTs technology, Sigma Genosys). A standard Westernblot analysis was employed to determine which peptides cross-react withG9 (FIG. 15). The cellulose membrane was wetted in methanol and blockedin Blocking Solution (5% BSA, 1×TBS, 0.1% Tween-20, 0.1% NaN₃) overnightat 4° C. Fresh Blocking Solution containing 1 mg/ml purified 10G9antibody was added, and the blot was incubated overnight at 4° C. Theblot was washed three times, 5 min each, in TBS-T (1×TBS, 0.1%Tween-20), and incubated for one hour in a 1:10,000 dilution ofHRP-conjugated goat anti-human IgG (H+L) (Jackson ImmunoResearch cat.109-035-088) in diluent (5% BSA, 1×TBS, 0.1% Tween-20). The blot wasdeveloped using SuperSignal West Femto ECL Substrate Kit (Pierce cat.34095), followed by a one second exposure to BioMAX film (Kodak). Bythis method, peptides #6, 13, 14, 15, and possibly peptide #23 werespecifically recognized by the G9 antibody, which correspond to SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:47,respectively.

The present invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

1. An isolated human monoclonal antibody that specifically binds tohuman GM-CSF wherein the heavy chain comprises the amino acid sequenceof SEQ ID NO:
 8. 2. The antibody of claim 1, wherein the light chaincomprises the amino acid sequence of SEQ ID NO:12.
 3. The antibody ofclaim 1, wherein the light chain comprises the amino acid sequence ofSEQ ID NO:
 18. 4. The antibody of claim 1, comprising two heavy chains.5. The antibody of claim 1, comprising two light chains.
 6. A method ofmaking the antibody of claim 1 comprising culturing a hybridoma underconditions suitable to produce the antibody, and recovering the antibodyfrom the cell culture.